Innovative discovery of therapeutic, diagnostic, and antibody compositions related to protein fragments of tyrosyl-trna synthetases

ABSTRACT

Provided are compositions comprising newly identified protein fragments of aminoacyl-tRNA synthetases, polynucleotides that encode them and complements thereof, related agents, and methods of use thereof in diagnostic, drug discovery, research, and therapeutic applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.14/684,052, filed Apr. 10, 2015; which is a Continuation of U.S.application Ser. No. 13/818,929, filed May 21, 2013; which is a U.S.national phase of International Patent Application No.PCT/US2011/049223, filed Aug. 25, 2011; which claims the benefit under35 U.S.C. §119(e) of U.S. provisional patent application No. 61/377,006filed on Aug. 25, 2010, and U.S. provisional patent application No.61/488,616 filed on May 20, 2011, the entire contents of each of which,are incorporated herein by reference.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is ATYR 089 04US ST25.txt. The text file is about64 KB, was created on Jul. 21, 2016, and is being submittedelectronically via EFS-Web.

TECHNICAL FIELD

The present invention relates generally to compositions comprising newlyidentified protein fragments of aminoacyl-tRNA synthetases and otherproteins, polynucleotides that encode them and complements thereof,related agents, and methods of use thereof in diagnostic, drugdiscovery, research, and therapeutic applications.

BACKGROUND

For over four decades, aminoacyl-tRNA synthetases (AARSs) were thoughtof as essential housekeeping proteins that catalyze the aminoacylationof tRNA molecules as part of the decoding of genetic information duringthe process of protein translation. AARSs have been extensively studiedin this respect, and many of their full-length sequences were cloned forsequence analysis and to provide a rich source of biochemicalexperimentation. Some fragments of AARSs, and other proteins, however,possess unexpected activities not associated with aminoacylation,including extracellular signaling activities that modulate pathwaysbeyond protein translation. Generally, these unexpected activities arenot observed in the context of the full-length or parental proteinsequences; instead, they are observed following removal or resection ofAARS protein fragments from their parental sequences, or by expressingand sufficiently purifying fragment AARS sequences and then testing fornovel, non-synthetase related activities.

While the full-length sequences of AARS have been known for some time,no systematic experimental analysis has been conducted to elucidate suchAARS protein fragments, or protein fragments from related or associatedproteins, or to evaluate the potential role of the full length AARSproteins for novel biological activities outside of the context of aminoacid synthesis. In portions of this specification, such AARS proteinfragments, AARS domains, or AARS alternative splice variants arereferred to herein as “resectins”. In its broadest context, the term“resectin” refers to a portion of a protein which has been excised orrestricted (either by means of proteolysis, alternative splicing,mutagenesis, or recombinant genetic engineering) from the context of itsnative full-length or parental protein sequence, which often otherwisemasks its novel biological activities. Likewise, no systematicexperimental analysis has been conducted to explore the use of suchresectins as biotherapeutic agents, diagnostic agents, or drug targetsin the treatment of various medical conditions, or their potentialassociation with human diseases. As essential housekeeping genes with aknown function in mammals that is critical to life, AARSs were neitherconsidered as drug targets in mammals, nor were they parsed out bystandard genomic sequencing, bioinformatic, or similar efforts toidentify resectins having non-synthetase activities. Standardbiochemical research efforts have similarly been directed away fromcharacterizing the biological properties of AARS resectins and theirpotential therapeutic and diagnostic relevance, mainly due to thepreviously understood role of their corresponding full-length parentalAARSs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the domain structure of the Tyrosyl aminoacyl tRNAsynthetase overlaid with the relative positions and sizes of theC-terminal AARS polypeptides identified from mass spectrometry analysis.

FIG. 2 shows the domain structure of the Tyrosyl aminoacyl tRNAsynthetase overlaid with the relative positions and sizes of theC-terminal AARS polypeptides identified from deep sequencing oftranscriptomes.

BRIEF SUMMARY OF THE INVENTION

Embodiments of the present invention relate generally to the discoveryof protein fragments of aminoacyl-tRNA synthetases (AARSs), whichpossess non-canonical biological activities, such as extracellularsignaling activities, and/or other characteristics of therapeutic anddiagnostic relevance. The AARSs are universal and essential elements ofthe protein synthesis machinery found in all organisms, but human AARSsand their associated proteins have naturally-occurring resectedvariants, with potent cell signaling activities that contribute tonormal functioning of humans. The activities of these protein fragmentsare distinct from the protein synthesis activities commonly known forAARSs, and the present invention includes the discovery and developmentof these resected proteins as new biotherapeutic agents, new discoveryresearch reagents, and as new antigens/targets for directed biologicsand diagnostic agents that can be used to potentially treat or diagnosea wide variety of human diseases, such as inflammatory, hematological,neurodegenerative, autoimmune, hematopoietic, cardiovascular, andmetabolic diseases or disorders.

The AARS protein fragment(s) of the present invention may therefore bereferred to as “resectins,” or alternatively as “appendacrines.” Asnoted above, the term “resectin” derives from the process of excising orresecting a given AARS protein fragment from the context of itsfull-length parent AARS sequence, which typically masks itsnon-canonical activities. In certain instances, the AARS proteinfragments and polynucleotides of the present invention were identifiedthrough the occurrence of this resection process, whethernaturally-occurring (e.g., proteolytic, splice variant),artificially-induced, or predicted. The term “appendacrine” derives froma combination of “append” (from Latin—appender) and to “separate” or“discern” (from Greek—crines),” and also reflects the separation of oneor more appended domains of the AARS protein fragments from theircorresponding full-length or parent AARS sequences.

Although a few AARS fragments have been previously shown to havenon-synthetase activities, the expression, isolation, purification, andcharacterization of such fragments for biotherapeutic, discovery, ordiagnostic utility is limited, and persons skilled in the art would nothave readily appreciated such activities to associate with each memberof the entire family of AARSs, or with alternative fragments. Here, amethodical approach was utilized to discover and verify AARS proteinfragments for the 20 mitochondrial and 20 cytosolic AARSs (andassociated proteins) for biotherapeutic discovery and diagnosticutility. For instance, certain of the present AARS protein fragment(s)and polynucleotides that encode them are identified from biologicalsamples using mass spectrometry (MS), mainly to identify proteolyticfragments, and others were identified by deep sequencing techniques,mainly to identify splice variants. Other AARS protein fragment(s) areidentified using in silico predictions of amino acid sequences, such asby computationally comparing synthetases from humans and lower organismsalong with key demarcations (e.g., protease sites); this approachutilized sequence analysis of the full-length AARS based on specificcriteria to discern proteolytic fragments and functional domainspossessing non-canonical biological activities.

Novel resectins of the AARSs are unexpected, and their differentialexpression is also unexpected. Specific resections are typically seenunder different treatments (e.g., from cells grown in media with orwithout serum), at different stages of growth (e.g., adult brain vs.fetal brain) and for different tissue types (e.g., pancreas vs. liver).The pattern of expression is not the same for all aminoacyl tRNAsynthetases despite the fact that the canonical functions for allaminoacyl tRNA synthetases are needed in the same cell locations and inrelatively proportional amounts. One would not expect the levels of anaminoacyl tRNA synthetase activity to increase without an increase inthe amounts of other aminoacyl tRNA synthetase activities at the sametime. The mass spectrometry and deep sequencing data indicates thataminoacyl tRNA synthetase resectins do have varying levels and do occurin different sites and at different stages.

In addition, AARS protein fragments can be expressed and purified tosufficiently high purity to discern their biological properties.Previously, fragments were often not of sufficient purity, folding, andstability to enable proper biological characterization of non-synthetaseactivities. Cell based assays, for instance, are used in conjunctionwith sufficiently pure, stable, soluble and folded resectins to revealtheir important biotherapeutic, discovery or diagnostic activities.

In particular, embodiments of the present invention relate to proteinfragments of tyrosyl tRNA synthetases, related agents and compositionsof biotherapeutic, discovery, or diagnostic utility, and methods of usethereof. The compositions of the present invention are useful in avariety of diagnostic, drug discovery, and therapeutic applications, asdescribed herein. Preferably, the AARS proteins and fragments arepurified and stored in suitable condition to the extent required forsuch biotherapeutic, discovery, or diagnostic uses.

Certain embodiments include compositions, comprising an isolatedaminoacyl-tRNA synthetase (AARS) protein fragment of at least about 100,90, 80, 70, 60, 50 or 40 amino acids that comprises an amino acidsequence as set forth in Table(s) 1-3Table(s) 1-3, and has a solubilityof at least about 5 mg/ml, and wherein the composition has a purity ofat least about 95% on a protein basis, and less than about 10 EU/mgprotein endotoxin. In one aspect, the composition is a therapeuticcomposition. In specific embodiments, the composition is substantiallyserum free. In some embodiments the AARS protein fragment comprises anon-canonical activity. In some embodiments, the non-canonicalbiological activity is selected from modulation of extracellularsignaling, modulation of cell proliferation, modulation of celldifferentiation, modulation of gene transcription, modulation ofcytokine production or activity, modulation of cytokine receptoractivity, and modulation of inflammation. In some embodiments, the AARSprotein fragment has an EC₅₀ of less than about 1 nM, about 5 nM, about10 nM, about 50 nM, about 100 nM or about 200 nM for a cell-basednon-canonical biological activity.

In certain embodiments the AARS protein fragment is fused to aheterologous polypeptide. In some embodiments, the AARS fusion proteinsubstantially retains a non-canonical activity of the AARS proteinfragment. In some embodiments, the AARS fusion protein suppresses anon-canonical activity of the AARS protein fragment. In someembodiments, the heterologous polypeptide is attached to the N-terminusof the AARS protein fragment. In some embodiments, the heterologouspolypeptide is attached to the C-terminus of the AARS protein fragment.In one aspect of any of these embodiments the heterologous polypeptideis selected from the group consisting of purification tags, epitopetags, targeting sequences, signal peptides, membrane translocatingsequences, and PK modifiers.

In certain embodiments, the composition comprises an AARS proteinfragment at a concentration of least about 10 mg/mL. In certainembodiments the composition comprises an AARS protein fragment which isat least 90% monodisperse. In certain embodiments the compositioncomprises less than about 3% high molecular weight aggregated proteins.In certain embodiments the composition exhibits less than 3% aggregationwhen stored at a concentration of at least 10 mg/mL in PBS for one weekat 4° C. In certain embodiments the composition exhibits less than 3%aggregation when stored at a concentration of at least 10 mg/mL in PBSfor one week at room temperature.

Various assays for measuring such features of resectins are describedherein and may be used to define aspects of the invention. In certainaspects, these features will be preferable for biotherapeutic utility ofthe AARS protein fragments described herein.

Certain embodiments include compositions, comprising an isolatedaminoacyl-tRNA synthetase (AARS) protein fragment of at least 120 aminoacids that differs from an amino acid sequence set forth in Table(s) 1-3by substitution, deletion, and/or addition of about 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids, whereinthe altered protein fragment substantially retains a non-canonicalactivity of the unaltered protein, or has a dominant negative phenotypein relation to the non-canonical activity, wherein the protein fragmenthas a solubility of at least about 5 mg/ml, and wherein the compositionhas a purity of at least about 95% on a protein basis and less thanabout 10 EU/mg protein endotoxin. In specific embodiments, thecomposition is substantially serum free.

Other embodiments include compositions, comprising an isolated antibodythat specifically binds to an isolated aminoacyl-tRNA synthetase (AARS)protein fragment as set forth in Table(s) 1-3, wherein affinity of theantibody for the AARS protein fragment is about 10× stronger than itsaffinity for a corresponding full-length AARS polypeptide. One of thesurprising aspects of the present invention includes certain resectinspossessing “new” surfaces accessible to antibody or other directedbiologics, whereas the full length AARS “hides” or covers these surfaceswith other sequences or adjacent domains. The process of resecting canalso create greater aqueous accessibility for revealing previouslyunidentified biological activities. Some embodiments includecompositions, comprising an isolated antibody that specifically binds toan isolated aminoacyl-tRNA synthetase (AARS) protein fragment as setforth in Table(s) 1-3, wherein the antibody has an affinity of at leastabout 10 nM for the AARS protein fragment, and an affinity of at leastabout 100 nM for a corresponding full-length AARS polypeptide. In someembodiments, the antibody binds to an epitope located within an AARSpolypeptide unique splice junction as set forth in any of Table(s) 1-3,or to an amino acid sequence C-terminal of this splice site. In certainembodiments, the antibody antagonizes the non-canonical activity of theAARS protein fragment. Such antagonists may optionally bind thecorresponding parental or full-length AARS.

Other aspects relate to bioassay systems, comprising a substantiallypure aminoacyl-tRNA synthetase (AARS) protein fragment of at least 120amino acids that comprises an amino acid sequence as set forth inTable(s) 1-3, and a binding partner that binds to the AARS proteinfragment. In one aspect, the binding partner is selected from the groupconsisting of a cellular surface receptor protein, nucleic acid, lipidmembrane, cell regulatory protein, enzyme, and transcription factor.Optionally, such a receptor may be part of a cell, preferably a cellrelevant to the revealed biology of the resectin.

Certain embodiments include cellular compositions, comprising anisolated aminoacyl-tRNA synthetase (AARS) protein fragment of at least120 amino acids that comprises an amino acid sequence as set forth inTable(s) 1-3, and an engineered population of cells in which at leastone cell comprises a polynucleotide encoding said AARS protein fragment.In one aspect, the cells are capable of growing in a serum free medium.

Also included are detection systems, comprising a substantially pureaminoacyl-tRNA synthetase (AARS) protein fragment of at least 50 or 100amino acids that comprises an amino acid sequence as set forth inTable(s) 1-3, a cell that comprises a cell-surface receptor or anextracellular portion thereof that binds to the protein fragment, and amolecule of less than about 2000 daltons, or a second polypeptide, whichmodulates binding or interaction between the AARS protein fragment andthe extracellular receptor.

Particular embodiments include diagnostic systems, comprising asubstantially pure aminoacyl-tRNA synthetase (AARS) protein fragment ofat least 120 amino acids that comprises an amino acid sequence as setforth in Table(s) 1-3, and a cell that comprises a cell-surface receptoror an extracellular portion thereof that binds to the AARS proteinfragment, wherein the system or cell comprises an indicator moleculethat allows detection of a change in the levels or activity of thecell-surface receptor or extracellular portion thereof.

Certain embodiments include cellular growth devices, comprising anisolated aminoacyl-tRNA synthetase (AARS) protein fragment of at least120 amino acids that comprises an amino acid sequence as set forth inTable(s) 1-3, an engineered population of cells in which at least onecell comprises a polynucleotide encoding said AARS protein fragment, atleast about 10 liters of serum-free cell media, and a sterile container.In specific embodiments, the cells utilized for any of the methods orcompositions described herein are capable of growing in serum-freemedia, optionally with an antibiotic and an inducer.

Some embodiments relate to antisense or RNA interference (RNAi) agents,comprising a sequence that is targeted against a unique splice junctionof an AARS splice variant as set forth in Table(s) 1-3.

Also included are therapeutic compositions, comprising an isolatedaminoacyl-tRNA synthetase (AARS) protein fragment of at least 120 aminoacids that comprises an amino acid sequence as set forth in Table(s)1-3, wherein the protein fragment specifically binds to a bindingpartner and has a solubility of at least about 5 mg/ml, and wherein thecomposition has a purity of at least about 95% on a protein basis. Insome aspects, the composition may have less than 10 EU endotoxin/mgprotein.

Also included are compositions, comprising an isolated aminoacyl-tRNAsynthetase (AARS) protein fragment of at least 120 amino acids that isat least 80%, 85%, 90%, 95%, 98%, or 100% identical to an amino acidsequence set forth in Table(s) 1-3, wherein the protein fragment has asolubility of at least about 5 mg/ml, and wherein the composition has apurity of at least about 95% on a protein basis and less than 10 EUendotoxin/mg protein. In any of these embodiments, the compositions maycomprise an AARS protein fragment that is at least about 50%, about 60%,about 70%, about 80%, about 90% or about 95% monodisperse with respectto its apparent molecular mass. In another aspect of any of theseembodiments, the compositions comprise less than about 10% (on a proteinbasis) high molecular weight aggregated proteins, or less than about 5%high molecular weight aggregated proteins, or less than about 4% highmolecular weight aggregated proteins, or less than about 3% highmolecular weight aggregated proteins, or less than 2% high molecularweight aggregated proteins, or less than about 1% high molecular weightaggregated proteins.

In another aspect of any of these embodiments, the compositions exhibitsless than about 10% aggregation when stored at a concentration of atleast 10 mg/mL in PBS for one week at 4° C., or less than about 5%aggregation when stored at a concentration of at least 10 mg/mL in PBSfor one week at 4° C., or less than about 3% aggregation when stored ata concentration of at least 10 mg/mL in PBS for one week at 4° C., orless than about 2% aggregation when stored at a concentration of atleast 10 mg/mL in PBS for one week at 4° C., or less than about 1%aggregation when stored at a concentration of at least 10 mg/mL in PBSfor one week at 4° C.

Certain embodiments include compositions, comprising a substantiallypure aminoacyl-tRNA synthetase (AARS) protein fragment of at least 120amino acids that comprises an amino acid sequence as set forth inTable(s) 1-3, and at least one covalently or non-covalently moietyattached thereto. In some embodiments, the moiety is a detectable label.In some embodiments, the moiety is a water soluble polymer. In someembodiments, the moiety is PEG. In one aspect of any of theseembodiments, the moiety is attached to the N-terminus of the proteinfragment. In one aspect of any of these embodiments, the moiety isattached to the C-terminus of the protein fragment.

Particular embodiments include compositions, comprising a solidsubstrate attached to an isolated aminoacyl-tRNA synthetase (AARS)protein fragment of at least 120 amino acids that comprises an aminoacid sequence as set forth in Table(s) 1-3, or a biologically activefragment or variant thereof, wherein the protein fragment has asolubility of at least about 5 mg/ml, and the composition has a purityof at least about 95% on a protein basis.

Also included are compositions, comprising a binding agent thatspecifically binds to an isolated aminoacyl-tRNA synthetase (AARS)protein fragment as set forth in Table(s) 1-3, wherein the binding agenthas an affinity of at least about 1 nM for the protein fragment. In oneaspect, the binding agent binds to an epitope located within an AARSpolypeptide unique splice junction as set forth in any of Table(s) 1-3,or to an amino acid sequence C-terminal of this splice site. In someembodiments, the binding agent antagonizes a non-canonical activity ofthe AARS polypeptide.

Certain embodiments include isolated aminoacyl-tRNA synthetase (AARS)polypeptides, comprising an amino acid sequence of an AARS proteinfragment as described herein, an amino acid sequence encoded by an AARSpolynucleotide as described herein, or a variant or fragment thereof.Certain AARS polypeptides comprise an amino acid sequence that is atleast 80%, 85%, 90%, 95%, 98%, or 100% identical to an AARS referencesequence as disclosed in Table(s) 1-3, or Table E2. Certain AARSpolypeptides consist essentially of an amino acid sequence that is atleast 80%, 85%, 90%, 95%, 98%, or 100% identical to an AARS referencesequence as disclosed in Table(s) 1-3, or Table E2. In certainembodiments, the polypeptide comprises a non-canonical biologicalactivity. In specific embodiments, the non-canonical biological activityis selected from modulation of cell signaling (e.g., extracellularsignaling), modulation of cell proliferation, modulation of cellmigration, modulation of cell differentiation, modulation of apoptosisor cell death, modulation of angiogenesis, modulation of cell binding,modulation of cellular metabolism, modulation of cellular uptake,modulation of gene transcription, or secretion, modulation of cytokineproduction or activity, modulation of cytokine receptor activity, andmodulation of inflammation.

Other aspects include antibodies and other binding agents that exhibitbinding specificity for an isolated AARS polypeptide as describedherein, a binding partner of the AARS polypeptide, or the complex ofboth. In some embodiments, the affinity of the antibody or binding agentfor the AARS polypeptide is about 10× stronger than its affinity for acorresponding full-length AARS polypeptide. In specific embodiments, thebinding agent is selected from a peptide, peptide mimetic, an adnectin,an aptamer, and a small molecule. In certain embodiments, the antibodyor binding agent antagonizes a non-canonical activity of the AARSpolypeptide. In other embodiments, the antibody or binding agentagonizes a non-canonical activity of the AARS polypeptide.

Certain embodiments include isolated aminoacyl-tRNA synthetase (AARS)polynucleotides, comprising a nucleotide sequence of an AARSpolynucleotide as described herein, a nucleotide sequence that encodesan AARS protein fragment as described herein, or a variant, a fragment,or a complement thereof. Certain AARS polynucleotides comprise anucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, or 100%identical to an AARS reference polynucleotide, or a complement thereof,as disclosed in Table(s) 1-3, or Table E2. In some embodiments, thenucleotide sequence is codon optimized for bacterial expression. In oneaspect, the nucleotide sequence is at least 80% identical apolynucleotide sequence disclosed in Table E2.

Specific AARS polynucleotides consist essentially of a nucleotidesequence that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical toan AARS reference polynucleotide, or a complement thereof, as disclosedin Table(s) 1-3, or Table E2. Other AARS polynucleotides comprise orconsist essentially of a nucleotide sequence that specificallyhybridizes to an AARS reference polynucleotide, as disclosed in Table(s)1-3, or Table E2. In certain embodiments, the polynucleotide is selectedfrom a primer, a probe, and an antisense oligonucleotide. In specificembodiments, the primer, probe, or antisense oligonucleotide is targetedto a specific or unique splice junction, and/or sequence 3′ of thissplice site within an AARS polynucleotide.

Certain embodiments include methods of determining presence or levels ofan AARS protein fragment in a sample, comprising contacting the samplewith one or more binding agents that specifically bind to an AARSprotein fragment as described herein, detecting the presence or absenceof the binding agent, and thereby determining the presence or levels ofthe AARS protein fragment. Other embodiments include methods ofdetermining presence or levels of an AARS protein fragment in a sample,comprising analyzing the sample with a detector that is capable ofspecifically identifying a protein fragment as described herein, andthereby determining the presence or levels of the AARS protein fragment.In specific embodiments, the detector is a mass spectrometer (MS), aflow cytometer, a protein imaging device, an enzyme-linked immunosorbentassays (ELISA), or a protein microarray. Certain embodiments comprisecomparing the presence or levels of the AARS protein fragment to acontrol sample or a predetermined value. Certain embodiments comprisecharacterizing the state of the sample to distinguish it from thecontrol. In specific embodiments, the sample and control comprise a cellor tissue, and the method comprises distinguishing between cells ortissues of different species, cells of different tissues or organs,cells at different cellular developmental states, cells at differentcellular differentiation states, cells at different physiologicalstates, or healthy and diseased cells. For instance, selected resectinsmay be more abundant under conditions such as stress or insult.

Certain embodiments include discovery methods of, and relatedcompositions for, identifying a compound that specifically binds to anaminoacyl-tRNA synthetase (AARS) polypeptide as described herein, or oneor more of its cellular binding partners, comprising a) combining theAARS polypeptide or its cellular binding partner or both with at leastone test compound under suitable conditions, and b) detecting binding ofthe AARS polypeptide or its cellular binding partner or both to the testcompound, thereby identifying a compound that specifically binds to theAARS polypeptide or its cellular binding partner or both. In certainembodiments, the test compound is a polypeptide or peptide, an antibodyor antigen-binding fragment thereof, a peptide mimetic, or a smallmolecule. In certain embodiments, the test compound agonizes anon-canonical biological activity of the AARS polypeptide or itscellular binding partner. In other embodiments, the test compoundantagonizes a non-canonical biological activity of the AARS polypeptideor its cellular binding partner. Certain embodiments include a compoundidentified by the above-method, such as an agonist (e.g., smallmolecule, peptide).

Certain embodiments include methods of determining presence or levels ofa polynucleotide sequence of an AARS splice variant in a sample,comprising contacting the sample with one or more oligonucleotides thatspecifically hybridize to an AARS polynucleotide as described herein,detecting the presence or absence of the oligonucleotides in the sample,and thereby determining the presence or levels of the polynucleotidesequence of the AARS splice variant. Other embodiments include methodsof determining presence or levels of a polynucleotide sequence of anAARS splice variant in a sample, comprising contacting the sample withat least two oligonucleotides that specifically amplify an AARSpolynucleotide as described herein, performing an amplificationreaction, detecting the presence or absence of an amplified product, andthereby determining presence or levels of the polynucleotide sequence ofthe AARS splice variant. In specific embodiments, the oligonucleotide(s)specifically hybridize to or specifically amplify a splice junction thatis unique to the AARS splice variant. Certain embodiments includecomparing the presence or levels of the AARS protein fragment or splicevariant to a control sample or a predetermined value. Certainembodiments include characterizing the state of the sample todistinguish it from the control. In specific embodiments, the sample andcontrol comprise a cell or tissue, and the method comprisesdistinguishing between cells or tissues of different species, cells ofdifferent tissues or organs, cells at different cellular developmentalstates, cells at different cellular differentiation states, or healthyand diseased cells.

Some embodiments include pharmaceutical compositions, comprising an AARSpolynucleotide described herein, an AARS polypeptide described herein, abinding agent as described herein, or a compound identified by theabove-method or described herein, and a pharmaceutically acceptableexcipient or carrier.

Certain embodiments include methods of modulating a cellular activity ofa cell, comprising contacting the cell with an AARS polynucleotidedescribed herein, an AARS polypeptide described herein, a binding agentdescribed herein, a compound of the above-method or described herein, ora pharmaceutical composition described herein. In specific embodiments,the cellular activity is selected from cell proliferation, cellmigration, cell differentiation, apoptosis or cell death, cellsignaling, angiogenesis, cell binding, cellular uptake, cell secretion,metabolism, cytokine production or activity, cytokine receptor activity,gene transcription, and inflammation. In one aspect, the cell isselected from the group consisting of pre-adipocytes, bone marrow,neutrophils, blood cells, hepatocytes, astrocytes, mesenchymal stemcells, and skeletal muscle cells.

In certain embodiments, the cell is in a subject. Certain embodimentscomprise treating the subject, wherein the subject has a conditionassociated with a neoplastic disease, an immune system disease orcondition, an infectious disease, a metabolic disease, an inflammatorydisorder, neuronal/neurological disease, a muscular/cardiovasculardisease, a disease associated with aberrant hematopoiesis, a diseaseassociated with aberrant angiogenesis, or a disease associated withaberrant cell survival.

Also included are processes for manufacturing a pharmaceutical compound,comprising: a) performing an in vitro screen of one or more candidatecompounds in the presence an AARS protein fragment of at least 120 aminoacids that comprises an amino acid sequence as set forth in Table(s)1-3, to identify a compound that specifically binds to the AARS proteinfragment; b) performing a cell-based or biochemical or receptor assaywith the compound identified in step a), to identify a compound thatmodulates one or more non-canonical activities of the AARS proteinfragment; c) optionally assessing the structure-activity relationship(SAR) of the compound identified in step b), to correlate its structurewith modulation of the non-canonical activity, and optionallyderivatizing the compound to alter its ability to modulate thenon-canonical activity; and d) producing sufficient amounts of thecompound identified in step b), or the derivatized compound in step c),for use in humans, thereby manufacturing the pharmaceutical compound.

Other embodiments include processes for manufacturing a pharmaceuticalcompound, comprising: a) performing an in vitro screen of one or morecandidate compounds in the presence a cell-surface receptor or anextracellular portion thereof that specifically binds to an AARS proteinfragment of Table(s) 1-3, to identify a compound that specifically bindsto the cell-surface receptor or extracellular portion thereof; b)performing a cell-based or biochemical or receptor assay with thecompound identified in step a), to identify a compound that modulatesone or more non-canonical activities of the AARS protein fragment; c)optionally assessing the structure-activity relationship (SAR) of thecompound identified in step b), to correlate its structure withmodulation of the non-canonical activity, and optionally derivatizingthe compound to alter its ability to modulate the non-canonicalactivity; and d) producing sufficient amounts of the compound identifiedin step b), or the derivatized compound in step c), for use in humans,thereby manufacturing the pharmaceutical compound.

Some embodiments include a cellular composition, comprising anengineered population of cells in which at least one cell comprises apolynucleotide encoding a heterologous full length aminoacyl-tRNAsynthetase (AARS) protein, wherein the cells are capable of growing in aserum-free medium. In one aspect, the full length aminoacyl-tRNAsynthetase (AARS) protein comprises a heterologous purification orepitope tag to facilitate purification of an AARS protein fragment. Inanother aspect, the full length aminoacyl-tRNA synthetase (AARS) proteincomprises a heterologous proteolysis site to enable production of theAARS protein fragment upon cleavage.

Some embodiments include a method for producing an AARS polypeptide asset forth in Table(s) 1-3, or Table E2 in situ within a cell,comprising; i) expressing a heterologous full length aminoacyl-tRNAsynthetase (AARS) protein within the cell, wherein the cell comprises aprotease capable of cleaving the heterologous full length aminoacyl-tRNAsynthetase (AARS) protein to produce the AARS polypeptide.

Some embodiments include a method for producing an AARS polypeptide asset forth in Table(s) 1-3, or Table E2 comprising contacting an isolatedfull length aminoacyl-tRNA synthetase (AARS) protein with a proteasethat is capable of cleaving the full length aminoacyl-tRNA synthetase(AARS) protein and producing an AARS polypeptide.

Some embodiments include an engineered full length aminoacyl-tRNAsynthetase (AARS) protein comprising a heterologous proteolysis site toenable the proteolytic generation of an AARS protein fragment as setforth in any of Table(s) 1-3 or Table E2.

Some embodiments include a composition, comprising an isolated fulllength aminoacyl-tRNA synthetase protein, wherein the composition has apurity of at least about 95% on a protein basis, less than about 10 EUendotoxin/mg protein, and is substantially serum free. In one aspect,the full length aminoacyl-tRNA synthetase protein is present at aconcentration of at least 10 mg/mL, and is at least 90% monodisperse.

A further embodiment includes a method of treating a disease or disordermediated by the dysregulation of the expression, activity orspatiotemporal location of a tRNA synthetase via the administration ofan AARS protein fragment, or nucleic acid encoding the ARRS proteinfragment, as set forth in any of Table(s) 1-3, or Table E2. In oneaspect of this embodiment, the disease is selected from the groupconsisting of cancer, neuropathy, diabetes, and inflammatory disorders.

In particular embodiments, the disease is Charcot-Marie-Tooth DiseaseType 2D (CMT2D) or Distal Spinal Muscular Atrophy Type V (dSMA-V). Inspecific embodiments, the disease is CMT2D, and the at least one symptomincludes reduced nerve conduction velocity. In some embodiments, thedisease is dSMA-V. In certain embodiments, the at least one symptom ismuscle weakness.

As noted above, also included are fusion proteins that comprise an AARSprotein fragment of any one or more of Tables 1-3 and a heterologousfusion partner. In certain embodiments, the AARS fusion proteinsubstantially retains a non-canonical activity of the AARS proteinfragment. In some embodiments, the AARS fusion protein suppresses anon-canonical activity of the AARS protein fragment. In particularembodiments, the heterologous polypeptide is attached to the N-terminusof the AARS protein fragment. In some embodiments, the heterologouspolypeptide is attached to the C-terminus of the AARS protein fragment.In specific embodiments, the heterologous polypeptide is selected fromthe group consisting of purification tags, epitope tags, targetingsequences, signal peptides, membrane translocating sequences, and PKmodifiers.

Also included are methods of reducing or ameliorating a symptom of adisease mediated by a mutation, inappropriate subcellular association,over-expression, and/or subcellular distribution of a tyrosyl-tRNAsynthetase (TyrRS), comprising administering to a subject an isolatedantibody or binding agent that specifically binds to an isolatedaminoacyl-tRNA synthetase (AARS) protein fragment as set forth inTable(s) 1-3, wherein affinity of the antibody for the AARS proteinfragment is at least about 10× stronger than its affinity for acorresponding full-length AARS polypeptide. In particular embodiments,the antibody or binding agent antagonizes a non-canonical activity ofthe AARS polypeptide

DETAILED DESCRIPTION OF THE INVENTION

TABLE OF CONTENTS

I. OVERVIEW . . . 15

II. DEFINITIONS . . . 16

III. PURIFIED AARS PROTEIN FRAGMENTS AND VARIANTS . . . 28

IV. AARS POLYNUCLEOTIDES . . . 61

V. ANTIBODIES . . . 74

VI. ANTIBODY ALTERNATIVES AND OTHER BINDING AGENTS . . . 79

VII. BIOASSAYS AND ANALYTICAL ASSAYS . . . 84

VIII. EXPRESSION AND PURIFICATION SYSTEMS . . . 86

IX. DIAGNOSTIC METHODS AND COMPOSITIONS . . . 99

X. ANTISENSE AND RNAI AGENTS . . . 115

-   -   A. ANTISENSE AGENTS . . . 116    -   B. RNA INTERFERENCE AGENTS . . . 124

XI. DRUG DISCOVERY . . . 132

XII. METHODS OF USE . . . 141

XIII. PHARMACEUTICAL FORMULATIONS, ADMINISTRATION AND KITS . . . 148

XIV. EXAMPLES . . . 157

I. OVERVIEW

The current invention is directed, at least in part, to the discovery ofnovel AARS polypeptides, and methods for their preparation and use,which represent the transformation of native wild type proteins into newforms that exhibit markedly different characteristics compared to thenaturally occurring full length tyrosyl tRNA synthetase genes. Such AARSpolypeptides were identified based on extensive sequence, and massspectrum analysis of expressed tyrosyl tRNA synthetases in differenttissues, followed by the systematic production and testing of eachpotential AARS polypeptide to identify protein sequences that representstable and soluble protein domains which exhibit novel biologicalactivities, and favorable therapeutic drug characteristics.

Based on this analysis at least two families of AARS polypeptidesderived from tyrosyl tRNA synthetase have been identified.

In one aspect, such tyrosyl RNA synthetase derived AARS polypeptidescomprise polypeptide sequences approximately comprising amino acids303-528 of the tyrosyl tRNA synthetase.

In a second aspect, such tyrosyl tRNA synthetase derived AARSpolypeptides comprise the alternatively spliced sequence comprisingamino acids 1-19+171-528 of tyrosyl tRNA synthetase.

These new AARS polypeptide families represent novel, previously unknownprotein products which exhibit inter alia i) novel biological activity,ii) favorable protein stability and aggregation characteristics, andiii) the ability to be expressed and produced at high level inprokaryotic expression systems, which are materially differentcharacteristics not found in the intact wild type protein.

II. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, preferred methods andmaterials are described. For the purposes of the present invention, thefollowing terms are defined below.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

By “about” is meant a quantity, level, value, number, frequency,percentage, dimension, size, amount, weight or length that varies by asmuch as 30, 25, 20, 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% to a referencequantity, level, value, number, frequency, percentage, dimension, size,amount, weight or length.

An “agonist” refers to a molecule that intensifies or mimics anactivity. For example, a non-canonical biological activity of an AARS,or another protein. Agonists may include proteins, nucleic acids,carbohydrates, small molecules, or any other compound or compositionthat modulates the activity of an AARS either by directly interactingwith the AARS or its binding partner, or by acting on components of thebiological pathway in which the AARS participates. Included are partialand full agonists.

As used herein, the term “amino acid” is intended to mean both naturallyoccurring and non-naturally occurring amino acids as well as amino acidanalogs and mimetics. Naturally occurring amino acids include the 20(L)-amino acids utilized during protein biosynthesis as well as otherssuch as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine,homocysteine, citrulline and ornithine, for example. Non-naturallyoccurring amino acids include, for example, (D)-amino acids, norleucine,norvaline, p-fluorophenylalanine, ethionine and the like, which areknown to a person skilled in the art. Amino acid analogs includemodified forms of naturally and non-naturally occurring amino acids.Such modifications can include, for example, substitution or replacementof chemical groups and moieties on the amino acid or by derivitizationof the amino acid. Amino acid mimetics include, for example, organicstructures which exhibit functionally similar properties such as chargeand charge spacing characteristic of the reference amino acid. Forexample, an organic structure which mimics Arginine (Arg or R) wouldhave a positive charge moiety located in similar molecular space andhaving the same degree of mobility as the e-amino group of the sidechain of the naturally occurring Arg amino acid. Mimetics also includeconstrained structures so as to maintain optimal spacing and chargeinteractions of the amino acid or of the amino acid functional groups.Those skilled in the art know or can determine what structuresconstitute functionally equivalent amino acid analogs and amino acidmimetics.

In certain aspects, the use of non-natural amino acids can be utilizedto modify (e.g., increase) a selected non-canonical activity of an AARSprotein fragment, or to alter the in vivo or in vitro half-life of theprotein. Non-natural amino acids can also be used to facilitate(selective) chemical modifications (e.g., pegylation) of an AARSprotein. For instance, certain non-natural amino acids allow selectiveattachment of polymers such as PEG to a given protein, and therebyimprove their pharmacokinetic properties.

Specific examples of amino acid analogs and mimetics can be founddescribed in, for example, Roberts and Vellaccio, The Peptides:Analysis, Synthesis, Biology, Eds. Gross and Meinhofer, Vol. 5, p. 341,Academic Press, Inc., New York, N.Y. (1983), the entire volume of whichis incorporated herein by reference. Other examples include peralkylatedamino acids, particularly permethylated amino acids. See, for example,Combinatorial Chemistry, Eds. Wilson and Czarnik, Ch. 11, p. 235, JohnWiley & Sons Inc., New York, N.Y. (1997), the entire book of which isincorporated herein by reference. Yet other examples include amino acidswhose amide portion (and, therefore, the amide backbone of the resultingpeptide) has been replaced, for example, by a sugar ring, steroid,benzodiazepine or carbo cycle. See, for instance, Burger's MedicinalChemistry and Drug Discovery, Ed. Manfred E. Wolff, Ch. 15, pp. 619-620,John Wiley & Sons Inc., New York, N.Y. (1995), the entire book of whichis incorporated herein by reference. Methods for synthesizing peptides,polypeptides, peptidomimetics and proteins are well known in the art(see, for example, U.S. Pat. No. 5,420,109; M. Bodanzsky, Principles ofPeptide Synthesis (1st ed. & 2d rev. ed.), Springer-Verlag, New York,N.Y. (1984 & 1993), see Chapter 7; Stewart and Young, Solid PhasePeptide Synthesis, (2d ed.), Pierce Chemical Co., Rockford, Ill. (1984),each of which is incorporated herein by reference). Accordingly, theAARS polypeptides of the present invention may be composed of naturallyoccurring and non-naturally occurring amino acids as well as amino acidanalogs and mimetics.

The term “antagonist” refers to a molecule that reduces or attenuates anactivity. For example, a non-canonical biological activity of an AARS,or another protein. Antagonists may include proteins such as antibodies,nucleic acids, carbohydrates, small molecules, or any other compound orcomposition that modulates the activity of an AARS or its bindingpartner, either by directly interacting with the AARS or its bindingpartner or by acting on components of the biological pathway in whichthe AARS participates. Included are partial and full antagonists.

The term “aminoacyl-tRNA synthetase” (AARS) refers generally to enzymesthat in their natural or wild-type form are capable of catalyzing theesterification of a specific amino acid or its precursor to one of allits compatible cognate tRNAs to form an aminoacyl-tRNA. In this“canonical” activity, aminoacyl-tRNA synthetases catalyze a two-stepreaction: first, they activate their respective amino acid by forming anaminoacyl-adenylate, in which the carboxyl of the amino acid is linkedin to the alpha-phosphate of ATP by displacing pyrophosphate, and then,when the correct tRNA is bound, the aminoacyl group of theaminoacyl-adenylate is transferred to the 2′ or 3′ terminal OH of thetRNA.

Class I aminoacyl-tRNA synthetases typically have two highly conservedsequence motifs. These enzymes aminoacylate at the 2′-OH of an adenosinenucleotide, and are usually monomeric or dimeric. Class IIaminoacyl-tRNA synthetases typically have three highly conservedsequence motifs. These enzymes aminoacylate at the 3′-OH of the sameadenosine, and are usually dimeric or tetrameric. The active sites ofclass II enzymes are mainly made up of a seven-stranded anti-parallelβ-sheet flanked by α-helices. Although phenylalanine-tRNA synthetase isclass II, it aminoacylates at the 2′-OH.

AARS polypeptides include sources of mitochondrial and cytoplasmic formsof tyrosyl-tRNA synthetase (TyrRS), a tryptophanyl-tRNA synthetase(TrpRS), a glutaminyl-tRNA synthetase (GlnRS), a glycyl-tRNA synthetase(GlyRS), a histidyl-tRNA synthetase (HisRS), a seryl-tRNA synthetase(SerRS), a phenylalanyl-tRNA synthetase (PheRS), an alanyl-tRNAsynthetase (AlaRS), an asparaginyl-tRNA synthetase (AsnRS), anaspartyl-tRNA synthetase (AspRS), a cysteinyl-tRNA synthetase (CysRS), aglutamyl-tRNA synthetase (GluRS), a prolyl-tRNA synthetase (ProRS), anarginyl-tRNA synthetase (ArgRS), an isoleucyl-tRNA synthetase (IleRS), aleucyl-tRNA synthetase (LeuRS), a lysyl-tRNA synthetase (LysRS), athreonyl-tRNA synthetase (ThrRS), a methionyl-tRNA synthetases (MetRS),or a valyl-tRNA synthetase (ValRS). The wild-type or parental sequencesof these AARS polypeptides are known in the art.

By “coding sequence” is meant any nucleic acid sequence that contributesto the code for the polypeptide product of a gene. By contrast, the term“non-coding sequence” refers to any nucleic acid sequence that does notcontribute to the code for the polypeptide product of a gene.

Throughout this specification, unless the context requires otherwise,the words “comprise,” “comprises,” and “comprising” will be understoodto imply the inclusion of a stated step or element or group of steps orelements but not the exclusion of any other step or element or group ofsteps or elements.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of” Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present. By “consisting essentially of” is meantincluding any elements listed after the phrase, and limited to otherelements that do not interfere with or contribute to the activity oraction specified in the disclosure for the listed elements. Thus, thephrase “consisting essentially of” indicates that the listed elementsare required or mandatory, but that other elements are optional and mayor may not be present depending upon whether or not they materiallyaffect the activity or action of the listed elements.

The recitation “endotoxin free” or “substantially endotoxin free”relates generally to compositions, solvents, and/or vessels that containat most trace amounts (e.g., amounts having no clinically adversephysiological effects to a subject) of endotoxin, and preferablyundetectable amounts of endotoxin. Endotoxins are toxins associated withcertain bacteria, typically gram-negative bacteria, although endotoxinsmay be found in gram-positive bacteria, such as Listeria monocytogenes.The most prevalent endotoxins are lipopolysaccharides (LPS) orlipo-oligo-saccharides (LOS) found in the outer membrane of variousGram-negative bacteria, and which represent a central pathogenic featurein the ability of these bacteria to cause disease. Small amounts ofendotoxin in humans may produce fever, a lowering of the blood pressure,and activation of inflammation and coagulation, among other adversephysiological effects.

Therefore, in pharmaceutical production of AARS polypeptides, it isoften desirable to remove most or all traces of endotoxin from drugproducts and/or drug containers, because even small amounts may causeadverse effects in humans. A depyrogenation oven may be used for thispurpose, as temperatures in excess of 300° C. are typically required tobreak down most endotoxins. For instance, based on primary packagingmaterial such as syringes or vials, the combination of a glasstemperature of 250° C. and a holding time of 30 minutes is oftensufficient to achieve a 3 log reduction in endotoxin levels. Othermethods of removing endotoxins are contemplated, including, for example,chromatography and filtration methods, as described herein and known inthe art. Also included are methods of producing AARS polypeptides in andisolating them from eukaryotic cells such as mammalian cells to reduce,if not eliminate, the risk of endotoxins being present in a compositionof the invention. Preferred are methods of producing AARS polypeptidesin and isolating them from serum free cells. Such compositionscomprising AARS polypeptides represent new formulations which exhibitnovel and new biological and therapeutic characteristics not found inAARS polypeptide compositions contaminated with serum or endotoxin whichhave the potential to bind to and alter the novel biological propertiesof the AARS polypeptides.

Endotoxins can be detected using routine techniques known in the art.For example, the Limulus Ameobocyte Lysate assay, which utilizes bloodfrom the horseshoe crab, is a very sensitive assay for detectingpresence of endotoxin, and reagents, kits and instrumentation for thedetection of endotoxin based on this assay are commercially available,for example from the Lonza Group. In this test, very low levels of LPScan cause detectable coagulation of the limulus lysate due a powerfulenzymatic cascade that amplifies this reaction. Endotoxins can also bequantitated by enzyme-linked immunosorbent assay (ELISA). To besubstantially endotoxin free, endotoxin levels may be less than about0.001, 0.005, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.08, 0.09, 0.1, 0.5,1.0, 1.5, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 EU/mg of protein.Typically, 1 ng lipopolysaccharide (LPS) corresponds to about 1-10 EU.

In certain embodiments, the “purity” of any given agent (e.g., AARSprotein fragment) in a composition may be specifically defined. Forinstance, certain compositions may comprise an agent that is at least80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% pure, includingall decimals in between, as measured, for example and by no meanslimiting, by high pressure liquid chromatography (HPLC), a well-knownform of column chromatography used frequently in biochemistry andanalytical chemistry to separate, identify, and quantify compounds.

As used herein, the terms “function” and “functional” and the like referto a biological, enzymatic, or therapeutic function.

By “gene” is meant a unit of inheritance that may occupy a specificlocus on a chromosome and consists of transcriptional and/ortranslational regulatory sequences and/or a coding region and/ornon-translated sequences (i.e., introns, 5′ and 3′ untranslatedsequences).

“Homology” refers to the percentage number of amino acids that areidentical or constitute conservative substitutions. Homology may bedetermined using sequence comparison programs such as GAP (Deveraux etal., 1984, Nucleic Acids Research 12, 387-395), which is incorporatedherein by reference. In this way sequences of a similar or substantiallydifferent length to those cited herein could be compared by insertion ofgaps into the alignment, such gaps being determined, for example, by thecomparison algorithm used by GAP.

The term “host cell” includes an individual cell or cell culture thatcan be or has been a recipient of any recombinant vector(s), isolatedpolynucleotide, or polypeptide of the invention. Host cells includeprogeny of a single host cell, and the progeny may not necessarily becompletely identical (in morphology or in total DNA complement) to theoriginal parent cell due to natural, accidental, or deliberate mutationand/or change. A host cell includes cells transfected or infected invivo or in vitro with a recombinant vector or a polynucleotide of theinvention. A host cell which comprises a recombinant vector of theinvention is a recombinant host cell.

By “isolated” is meant material that is substantially or essentiallyfree from components that normally accompany it in its native state. Forexample, an “isolated polynucleotide,” as used herein, includes apolynucleotide that has been purified from the sequences that flank itin its naturally-occurring state, e.g., a DNA fragment which has beenremoved from the sequences that are normally adjacent to the fragment.Alternatively, an “isolated peptide” or an “isolated polypeptide” andthe like, as used herein, includes the in vitro isolation and/orpurification of a peptide or polypeptide molecule from its naturalcellular environment, and from association with other components of thecell; i.e., it is not significantly associated with in vivo substances.

The term “mRNA” or sometimes refer by “mRNA transcripts” as used herein,include, but not limited to pre-mRNA transcript(s), transcriptprocessing intermediates, mature mRNA(s) ready for translation andtranscripts of the gene or genes, or nucleic acids derived from the mRNAtranscript(s). Transcript processing may include splicing, editing anddegradation. As used herein, a nucleic acid derived from an mRNAtranscript refers to a nucleic acid for whose synthesis the mRNAtranscript or a subsequence thereof has ultimately served as a template.A cDNA reverse transcribed from an mRNA, an RNA transcribed from thatcDNA, a DNA amplified from the cDNA, an RNA transcribed from theamplified DNA, etc., are all derived from the mRNA transcript anddetection of such derived products is indicative of the presence and/orabundance of the original transcript in a sample. Thus, mRNA derivedsamples include, but are not limited to, mRNA transcripts of the gene orgenes, cDNA reverse transcribed from the mRNA, cRNA transcribed from thecDNA, DNA amplified from the genes, RNA transcribed from amplified DNA,and the like.

“Non-canonical” activity as used herein, refers generally to either i) anew activity possessed by an AARS polypeptide of the invention that isnot possessed to any significant degree by the intact native full lengthparental protein, or ii) an activity that was possessed by the by theintact native full length parental protein, where the AARS polypeptideeither exhibits a significantly higher (i.e. at least 20% greater)specific activity compared to the intact native full length parentalprotein, or exhibits the activity in a new context; for example byisolating the activity from other activities possessed by the intactnative full length parental protein. In the case of AARS polypeptides,non-limiting examples of non-canonical activities include extracellularsignaling, RNA-binding, amino acid-binding, modulation of cellproliferation, modulation of cell migration, modulation of celldifferentiation (e.g., hematopoiesis, neurogenesis, myogenesis,osteogenesis, and adipogenesis), modulation of gene transcription,modulation of apoptosis or other forms of cell death, modulation of cellsignaling, modulation of cellular uptake, or secretion, modulation ofangiogenesis, modulation of cell binding, modulation of cellularmetabolism, modulation of cytokine production or activity, modulation ofcytokine receptor activity, modulation of inflammation, and the like.

The term “half maximal effective concentration” or “EC₅₀” refers to theconcentration of an AARS protein fragment, antibody or other agentdescribed herein at which it induces a response halfway between thebaseline and maximum after some specified exposure time; the EC₅₀ of agraded dose response curve therefore represents the concentration of acompound at which 50% of its maximal effect is observed. In certainembodiments, the EC₅₀ of an agent provided herein is indicated inrelation to a “non-canonical” activity, as noted above. EC₅₀ alsorepresents the plasma concentration required for obtaining 50% of amaximum effect in vivo. Similarly, the “EC₉₀” refers to theconcentration of an agent or composition at which 90% of its maximaleffect is observed. The “EC₉₀” can be calculated from the “EC₅₀” and theHill slope, or it can be determined from the data directly, usingroutine knowledge in the art. In some embodiments, the EC₅₀ of an AARSprotein fragment, antibody, or other agent is less than about 0.01,0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60,70, 80, 90, or 100 nM. Preferably, biotherapeutic composition will havean EC₅₀ value of about 1 nM or less.

The term “modulating” includes “increasing” or “stimulating,” as well as“decreasing” or “reducing,” typically in a statistically significant ora physiologically significant amount as compared to a control.Accordingly a “modulator” may be an agonist, an antagonist, or anymixture thereof depending upon the conditions used. An “increased” or“enhanced” amount is typically a “statistically significant” amount, andmay include an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30 or more times (e.g., 500, 1000 times) (including all integersand decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8,etc.) the amount produced by no composition (the absence of an agent orcompound) or a control composition. A “decreased” or reduced amount istypically a “statistically significant” amount, and may include a 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% decrease in the amount produced by nocomposition (the absence of an agent or compound) or a controlcomposition, including all integers in between. As one non-limitingexample, a control in comparing canonical and non-canonical activitiescould include the AARS protein fragment of interest compared to itscorresponding full-length AARS, or a fragment AARS having comparablecanonical activity to its corresponding full-length AARS. Other examplesof “statistically significant” amounts are described herein.

By “obtained from” is meant that a sample such as, for example, apolynucleotide extract or polypeptide extract is isolated from, orderived from, a particular source of the subject. For example, theextract can be obtained from a tissue or a biological fluid isolateddirectly from the subject. “Derived” or “obtained from” can also referto the source of a polypeptide or polynucleotide sequence. For instance,an AARS sequence of the present invention may be “derived” from thesequence information of an AARS proteolytic fragment or AARS splicevariant, or a portion thereof, whether naturally-occurring orartificially generated, and may thus comprise, consist essentially of,or consist of that sequence

The terms “polypeptide” and “protein” are used interchangeably herein torefer to a polymer of amino acid residues and to variants and syntheticand naturally occurring analogues of the same. Thus, these terms applyto amino acid polymers in which one or more amino acid residues aresynthetic non-naturally occurring amino acids, such as a chemicalanalogue of a corresponding naturally occurring amino acid, as well asto naturally-occurring amino acid polymers and naturally occurringchemical derivatives thereof. Such derivatives include, for example,post-translational modifications and degradation products includingpyroglutamyl, iso-aspartyl, proteolytic, phosphorylated, glycosylated,oxidatized, isomerized, and deaminated variants of the AARS referencefragment.

The recitations “sequence identity” or, for example, comprising a“sequence 50% identical to,” as used herein, refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity.

Terms used to describe sequence relationships between two or morepolynucleotides or polypeptides include “reference sequence,”“comparison window,” “sequence identity,” “percentage of sequenceidentity” and “substantial identity.” A “reference sequence” is at least12 but frequently 15 to 18 and often at least 25 monomer units,inclusive of nucleotides and amino acid residues, in length. Because twopolynucleotides may each comprise (1) a sequence (i.e., only a portionof the complete polynucleotide sequence) that is similar between the twopolynucleotides, and (2) a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window” refers to aconceptual segment of at least 6 contiguous positions, usually about 50to about 100, more usually about 100 to about 150 in which a sequence iscompared to a reference sequence of the same number of contiguouspositions after the two sequences are optimally aligned. The comparisonwindow may comprise additions or deletions (i.e., gaps) of about 20% orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by computerized implementations of algorithms (GAP, BESTFIT,FASTA, and TFASTA in the Wisconsin Genetics Software Package Release7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) orby inspection and the best alignment (i.e., resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected. Reference also may be made to the BLAST familyof programs as for example disclosed by Altschul et al., 1997, Nucl.Acids Res. 25:3389. A detailed discussion of sequence analysis can befound in Unit 19.3 of Ausubel et al., “Current Protocols in MolecularBiology,” John Wiley & Sons Inc, 1994-1998, Chapter 15.

Calculations of sequence similarity or sequence identity betweensequences (the terms are used interchangeably herein) are performed asfollows. To determine the percent identity of two amino acid sequences,or of two nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In certain embodiments, the length of a reference sequencealigned for comparison purposes is at least 30%, preferably at least40%, more preferably at least 50%, 60%, and even more preferably atleast 70%, 80%, 90%, 100% of the length of the reference sequence. Theamino acid residues or nucleotides at corresponding amino acid positionsor nucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position.

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch,(1970, J. Mol. Biol. 48: 444-453) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused unless otherwise specified) are a Blossum 62 scoring matrix with agap penalty of 12, a gap extend penalty of 4, and a frame shift gappenalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller (1989,Cabios, 4: 11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al., (1990, J. Mol. Biol, 215: 403-10). BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to nucleic acidmolecules of the invention. BLAST protein searches can be performed withthe XBLAST program, score=50, wordlength=3 to obtain amino acidsequences homologous to protein molecules of the invention. To obtaingapped alignments for comparison purposes, Gapped BLAST can be utilizedas described in Altschul et al., (1997, Nucleic Acids Res, 25:3389-3402). When utilizing BLAST and Gapped BLAST programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) can beused.

The term “solubility” refers to the property of an agent provided hereinto dissolve in a liquid solvent and form a homogeneous solution.Solubility is typically expressed as a concentration, either by mass ofsolute per unit volume of solvent (g of solute per kg of solvent, g perdL (100 mL), mg/ml, etc.), molarity, molality, mole fraction or othersimilar descriptions of concentration. The maximum equilibrium amount ofsolute that can dissolve per amount of solvent is the solubility of thatsolute in that solvent under the specified conditions, includingtemperature, pressure, pH, and the nature of the solvent. In certainembodiments, solubility is measured at physiological pH. In certainembodiments, solubility is measured in water or a physiological buffersuch as PBS. In certain embodiments, solubility is measured in abiological fluid (solvent) such as blood or serum. In certainembodiments, the temperature can be about room temperature (e.g., about20, 21, 22, 23, 24, 25° C.) or about body temperature (37° C.). Incertain embodiments, an agent such as an AARS protein fragment has asolubility of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, or 30 mg/ml at room temperature or at 37° C.

A “splice junction” as used herein includes the region in a mature mRNAtranscript or the encoded polypeptide where the 3′ end of a first exonjoins with the 5′ end of a second exon. The size of the region may vary,and may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100 or more (including all integers in between) nucleotide or amino acidresidues on either side of the exact residues where the 3′ end of oneexon joins with the 5′ end of another exon. An “exon” refers to anucleic acid sequence that is represented in the mature form of an RNAmolecule after either portions of a precursor RNA (introns) have beenremoved by cis-splicing or two or more precursor RNA molecules have beenligated by trans-splicing. The mature RNA molecule can be a messengerRNA or a functional form of a non-coding RNA such as rRNA or tRNA.Depending on the context, an exon can refer to the sequence in the DNAor its RNA transcript. An “intron” refers to a non-coding nucleic acidregion within a gene, which is not translated into a protein. Non-codingintronic sections are transcribed to precursor mRNA (pre-mRNA) and someother RNAs (such as long noncoding RNAs), and subsequently removed bysplicing during the processing to mature RNA.

A “splice variant” refers to a mature mRNA and its encoded protein thatare produced by alternative splicing, a process by which the exons ofthe RNA (a primary gene transcript or pre-mRNA) are reconnected inmultiple ways during RNA splicing. The resulting different mRNAs may betranslated into different protein isoforms, allowing a single gene tocode for multiple proteins.

A “subject,” as used herein, includes any animal that exhibits asymptom, or is at risk for exhibiting a symptom, which can be treated ordiagnosed with an AARS polynucleotide or polypeptide of the invention.Also included are subjects for which it is desirable to profile levelsof AARS polypeptides and/or polynucleotides of the invention, fordiagnostic or other purposes. Suitable subjects (patients) includelaboratory animals (such as mouse, rat, rabbit, or guinea pig), farmanimals, and domestic animals or pets (such as a cat or dog). Non-humanprimates and, preferably, human patients, are included.

“Treatment” or “treating,” as used herein, includes any desirable effecton the symptoms or pathology of a disease or condition that can beeffected by the non-canonical activities of an AARS polynucleotide orpolypeptide, as described herein, and may include even minimal changesor improvements in one or more measurable markers of the disease orcondition being treated. Also included are treatments that relate tonon-AARS therapies, in which an AARS sequence described herein providesa clinical marker of treatment. “Treatment” or “treating” does notnecessarily indicate complete eradication or cure of the disease orcondition, or associated symptoms thereof. The subject receiving thistreatment is any subject in need thereof. Exemplary markers of clinicalimprovement will be apparent to persons skilled in the art.

The practice of the present invention will employ, unless indicatedspecifically to the contrary, conventional methods of molecular biologyand recombinant DNA techniques within the skill of the art, many ofwhich are described below for the purpose of illustration. Suchtechniques are explained fully in the literature. See, e.g., Sambrook,et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2000); DNACloning: A Practical Approach, vol. I & II (D. Glover, ed.);Oligonucleotide Synthesis (N. Gait, ed., 1984); OligonucleotideSynthesis: Methods and Applications (P. Herdewijn, ed., 2004); NucleicAcid Hybridization (B. Hames & S. Higgins, eds., 1985); Nucleic AcidHybridization: Modern Applications (Buzdin and Lukyanov, eds., 2009);Transcription and Translation (B. Hames & S. Higgins, eds., 1984);Animal Cell Culture (R. Freshney, ed., 1986); Freshney, R. I. (2005)Culture of Animal Cells, a Manual of Basic Technique, 5^(th) Ed. HobokenN.J., John Wiley & Sons; B. Perbal, A Practical Guide to MolecularCloning (3^(rd) Edition 2010); Farrell, R., RNA Methodologies: ALaboratory Guide for Isolation and Characterization (3^(rd) Edition2005), Methods of Enzymology: DNA Structure Part A: Synthesis andPhysical Analysis of DNA Methods in Enzymology, Academic Press; UsingAntibodies: A Laboratory Manual: Portable Protocol NO. I by EdwardHarlow, David Lane, Ed Harlow (1999, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-544-7); Antibodies: A Laboratory Manual by Ed Harlow(Editor), David Lane (Editor) (1988, Cold Spring Harbor LaboratoryPress, ISBN 0-87969-3, 4-2), 1855. Handbook of Drug Screening, edited byRamakrishna Seethala, Prabhavathi B. Fernandes (2001, New York, N.Y.,Marcel Dekker, ISBN 0-8247-0562-9); and Lab Ref A Handbook of Recipes,Reagents, and Other Reference Tools for Use at the Bench, Edited JaneRoskams and Linda Rodgers, (2002, Cold Spring Harbor Laboratory, ISBN0-87969-630-3).

All publications, patents and patent applications cited herein arehereby incorporated by reference in their entirety.

III. PURIFIED AARS PROTEIN FRAGMENTS AND VARIANTS FOR THERAPEUTICS ANDOTHER APPLICATIONS

Surprisingly, and unlike their full-length parental sequences that areknown only for their aminoacylation-activities, it has been found thatAARS fragments possess biological activities important forbiotherapeutic, discovery and diagnostic applications. Embodiments ofthe present invention therefore include full length proteins, matureprotein isoforms and protein fragments of aminoacyl-tRNA synthetases(AARS), in addition to biologically active variants and fragmentsthereof. In certain embodiments, the proteins and fragments may arisethrough endogenous proteolysis, in vitro proteolysis, splice variation,or in silico prediction, among other mechanisms.

The AARS protein fragments described herein, and variants thereof, maypossess at least one “non-canonical” biological activity. The AARSprotein fragment(s) of the present invention are also referred to hereinas “AARS polypeptides” or “AARS reference polypeptides.” In certainembodiments, the AARS polypeptides provided herein comprise or consistessentially of all or a portion of the AARS polypeptide “referencesequence(s)” as set forth in Table(s) 1-3 below, which represent theamino acid sequence(s) of various fragments of tyrosyl tRNA synthetases.Mouse and human AARS protein sequences are highly related, typicallydiffering by no more than a few amino acids within an entire sequence, aparticular domain, or a particular protein fragment.

TABLE 1 AARS polypeptides identified by MS Type/ species/ SEQ.ID. NameResidues Amino acid and Nucleic Acid Sequences NO. TyrRS1^(C1) Protein/EKIDVGEAEPRTVVSGLVQFVPKEELQDRL SEQ.ID. Human/VVVLCNLKPQKMRGVESQGMLLCASIEGI NO. 12 390-528NRQVEPLDPPAGSAPGEHVFVKGYEKGQP DEELKPKKKVFEKLQADFKISEECIAQWKQTNFMTKLGSISCKSLKGGNIS TyrRS1^(C1) DNA/ GAGAAGATTGACGTGGGGGAAGCTGAACSEQ.ID. Human/ CACGGACTGTGGTGAGCGGCCTGGTACA NO. 13GTTCGTGCCCAAGGAGGAACTGCAGGAC AGGCTGGTAGTGGTGCTGTGCAACCTGAAACCCCAGAAGATGAGAGGAGTCGAGTC CCAAGGCATGCTTCTGTGTGCTTCTATAGAAGGGATAAACCGCCAGGTTGAACCTCT GGACCCTCCGGCAGGCTCTGCTCCTGGTGAGCACGTGTTTGTGAAGGGCTATGAAAA GGGCCAACCAGATGAGGAGCTCAAGCCCAAGAAGAAAGTCTTCGAGAAGTTGCAGG CTGACTTCAAAATTTCTGAGGAGTGCATCGCACAGTGGAAGCAAACCAACTTCATGA CCAAGCTGGGCTCCATTTCCTGTAAATCGCTGAAAGGGGGGAACATTAGCTAG TyrRS1^(C2) Protein/AYPDPSKQKPMAKGPAKNSEPEEVIPSRLD SEQ.ID. Human/IRVGKIITVEKHPDADSLYVEKIDVGEAEPR NO. 14 340-528TVVSGLVQFVPKEELQDRLVVVLCNLKPQ KMRGVESQGMLLCASIEGINRQVEPLDPPAGSAPGEHVFVKGYEKGQPDEELKPKKKVF EKLQADFKISEECIAQWKQTNFMTKLGSIS CKSLKGGNISTyrRS1^(C2) DNA/ GCCTACCCAGATCCCTCAAAGCAGAAGC SEQ.ID. Human/CAATGGCCAAAGGCCCTGCCAAGAATTC NO. 15 AGAACCAGAGGAGGTCATCCCATCCCGGCTGGATATCCGTGTGGGGAAAATCATCA CTGTGGAGAAGCACCCAGATGCAGACAGCCTGTATGTAGAGAAGATTGACGTGGGG GAAGCTGAACCACGGACTGTGGTGAGCGGCCTGGTACAGTTCGTGCCCAAGGAGGA ACTGCAGGACAGGCTGGTAGTGGTGCTGTGCAACCTGAAACCCCAGAAGATGAGAG GAGTCGAGTCCCAAGGCATGCTTCTGTGTGCTTCTATAGAAGGGATAAACCGCCAGG TTGAACCTCTGGACCCTCCGGCAGGCTCTGCTCCTGGTGAGCACGTGTTTGTGAAGG GCTATGAAAAGGGCCAACCAGATGAGGAGCTCAAGCCCAAGAAGAAAGTCTTCGAG AAGTTGCAGGCTGACTTCAAAATTTCTGAGGAGTGCATCGCACAGTGGAAGCAAACC AACTTCATGACCAAGCTGGGCTCCATTTCCTGTAAATCGCTGAAAGGGGGGAACATT AGCTAG

TABLE 1B TyrRS1^(C1)Mass spec peptides detected and inferred linking peptides Type/ SEQ.ID.species Sequence NO. Protein/ IDVGEAEPR SEQ.ID. mouse NO. 16 Protein/TVVSGLVQFVPKEELQDRLVVVLCNLKPQKMRGVDS SEQ.ID. mouse QGMLLCASVEGVSR NO. 17Protein/ QVEPLDPPAGSAPGER SEQ.ID. mouse NO. 18 Protein/VFVQGYEKGQPDEELKPKKKVFEKLQADFK SEQ.ID. mouse NO. 19 Protein/ ISEECIAQWKSEQ.ID. mouse NO. 20

TABLE 1C TyrRS1^(C1) Concatenated sequences based on mass spec peptidesdetected Type/ SEQ.ID. species Sequence NO. Protein/IDVGEAEPRTVVSGLVQFVPKEELQDRLVVVLCNLKPQ SEQ.ID. mouseKMRGVDSQGMLLCASVEGVSRQVEPLDPPAGSAPGER NO. 21VFVQGYEKGQPDEELKPKKKVFEKLQADFKISEECIAQ WK

TABLE 1D TyrRS1^(C1) Mass spec peptides detected and inferred linkingpeptides Type/ SEQ.ID. species Sequence NO. Protein/ SEPEEVIPSR SEQ.ID.mouse NO. 22 Protein/ LDIRVGKILSVEKHPDADSLYVEK SEQ.ID. mouse NO. 23Protein/ IDVGEAEPR SEQ.ID. mouse NO. 24 Protein/TVVSGLVQFVPKEELQDRLVVVLCNLKP SEQ.ID. mouse QKMR NO. 25 Protein/GVDSQGMLLCASVEGVSR SEQ.ID. mouse NO. 26 Protein/ QVEPLDPPAGSAPGERSEQ.ID. mouse NO. 27 Protein/ VFVQGYEK SEQ.ID. mouse NO. 28 Protein/GQPDEELKPKKKVFEKLQADFK SEQ.ID. mouse NO. 29 Protein/ ISEECIAQWK SEQ.ID.mouse NO. 30

TABLE 1E TyrRS1^(C1)Concatenated sequences based on mass spec peptides detected Type/SEQ.ID. species Sequence NO. Protein/SEPEEVIPSRLDIRVGKILSVEKHPDADSLYVEKIDVGE SEQ.ID. mouseAEPRTVVSGLVQFVPKEELQDRLVVVLCNLKPQKMRG NO. 31VDSQGMLLCASVEGVSRQVEPLDPPAGSAPGERVFVQGYEKGQPDEELKPKKKVFEKLQADFKISEECIAQWK

TABLE 2 AARS polypeptides and alternative transcripts identified by DeepSequencing Type/ species/ SEQ.ID Name ResiduesAmino acid and Nucleic Acid Sequences NO. TyRS1^(C3) Protein/MAKGPAKNSEPEEVIPSRLDIRVGKHTVEK SEQ.ID. Human/HPDADSLYVEKIDVGEAEPRTVVSGLVQFV NO. 32 350-528PKEELQDRLVVVLCNLKPQKMRGVESQG MLLCASIEGINRQVEPLDPPAGSAPGEHVFVKGYEKGQPDEELKPKJCKVFEKLQADFKI SEECIAQWKQTNFMTKLGSISCKSLKGGNI STyRS1^(C3) DNA/ ATGGCCAAAGGCCCTGCCAAGAATTCAG SEQ.ID. Human/AACCAGAGGAGGTCATCCCATCCCGGCT NO. 33 GGATATCCGTGTGGGGAAAATCATCACTGTGGAGAAGCACCCAGATGCAGACGCC TGTATGTAGAGAAGATTGACGTGGGGGAAGCTGAACCACGGACTGTGGTGAGCGGC CTGGTACAGTTCGTGCCCAAGGAGGAACTGCAGGACAGGCTGGTAGTGGTGCTGTG CAACCTGAAACCCCAGAAGATGAGAGGAGTCGAGTCCCAAGGCATGCTTCTGTGTGC TTCTATAGAAGGGATAAACCGCCAGGTTGAACCTCTGGACCCTCCGGCAGGCTCTGC TCCTGGTGAGCACGTGTTTGTGAAGGGCTATGAAAAGGGCCAACCAGATGAGGAGCT CAAGCCCAAGAAGAAAGTCTTCGAGAAGTTGCAGGCTGACTTCAAAATTTCTGAGGA GTGCATCGCACAGTGGAAGCAAACCAACTTCATGACCAAGCTGGGCTCCATTTCCTG TAAATCGCTGAAAGGGGGGAACATTAGC TAGTyrRS1^(C4) Protein/ MGDAPSPEEKLHLITRNLQVVHPGDLKNS SEQ.ID. Human/VEVALNKLLDPIREKFNTPALKKLASAAYP NO. 34 1-19 +DPSKQKPMAKGPAKNSEPEEVIPSRLDIRV 303-528 GKIITVEKHPDADSLYVEKIDVGEAEPRTVVSGLVQFVPKEELQDRLVVVLCNLKPQKM RGVESQGMLLCASIEGINRQVEPLDPPAGSAPGEHVFVKGYEKGQPDEELKPKKKVFEK LQADFKISEECIAQWKQTNFMTKLGSISCK SLKGGNISTyrRS1^(C4) DNA/ ATGGGGGACGCTCCCAGCCCTGAAGAGA SEQ.ID. Human/AACTGCACCTTATCACCCGGAACCTGCA NO. 35 GGTTGTACATCCTGGAGACCTGAAGAATTCTGTTGAAGTCGCACTGAACAAGTTGCT GGATCCAATCCGGGAAAAGTTTAATACCCCTGCCCTGAAAAAACTGGCCAGCGCTG CCTACCCAGATCCCTCAAAGCAGAAGCCAATGGCCAAAGGCCCTGCCAAGAATTCA GAACCAGAGGAGGTCATCCCATCCCGGCTGGATATCCGTGTGGGGAAAATCATCAC TGTGGAGAAGCACCCAGATGCAGACAGCCTGTATGTAGAGAAGATTGACGTGGGGG AAGCTGAACCACGGACTGTGGTGAGCGGCCTGGTACAGTTCGTGCCCAAGGAGGAA CTGCAGGACAGGCTGGTAGTGGTGCTGTGCAACCTGAAACCCCAGAAGATGAGAGG AGTCGAGTCCCAAGGCATGCTTCTGTGTGCTTCTATAGAAGGGATAAACCGCCAGGT TGAACCTCTGGACCCTCCGGCAGGCTCTGCTCCTGGTGAGCACGTGTTTGTGAAGGGC TATGAAAAGGGCCAACCAGATGAGGAGCTCAAGCCCAAGAAGAAAGTCTTCGAGAA GTTGCAGGCTGACTTCAAAATTTCTGAGGAGTGCATCGCACAGTGGAAGCAAACCAA CTTCATGACCAAGCTGGGCTCCATTTCCTGTAAATCGCTGAAAGGGGGGAACATTAG CTAG TyrRS1^(C5) Protein/MGDAPSPEEKLHLITRNLQALDEEYLKVD SEQ.ID. Human/AQFGGIDQRKIFTFAEKYLPALGYSKRVHL NO. 36 1-19 +MNPMVPGLTGSKMSSSEEESKIDLLDRKED 171-528 VKKKLKKAFCEPGNVENNGVLSFIKHVLFPLKSEFVILRDEKWGGNKTYTAYVDLEKDF AAEVVHPGDLKNSVEVALNKLLDPIREKFNTPALKKLASAAYPDPSKQKPMAKGPAKN SEPEEVIPSRLDIRVGKIITVEKHPDADSLYVEKIDVGEAEPRTVVSGLVQFVPKEELQDRL VVVLCNLKPQKMRGVESQGMLLCASIEGINRQVEPLDPPAGSAPGEHVFVKGYEKGQP DEELKPKKKVFEKLQADFKISEECIAQWKQTNFMTKLGSISCKSLKGGNIS TyrRS1^(C5) DNA/ ATGGGGGACGCTCCCAGCCCTGAAGAGASEQ.ID. Human/ AACTGCACCTTATCACCCGGAACCTGCA NO. 37GGCTTTGGATGAAGAGTATTTAAAAGTA GATGCCCAATTTGGAGGCATTGATCAGAGAAAGATTTTCACCTTTGCAGAGAAGTA CCTCCCTGCACTTGGCTATTCAAAACGGGTCCATCTGATGAATCCTATGGTTCCAGGA TTAACAGGCAGCAAAATGAGCTCTTCAGAAGAGGAGTCCAAGATTGATCTCCTTGA TCGGAAGGAGGATGTGAAGAAAAAACTGAAGAAGGCCTTCTGTGAGCCAGGAAATG TGGAGAACAATGGGGTTCTGTCCTTCATCAAGCATGTCCTTTTTCCCCTTAAGTCCGA GTTTGTGATCCTACGAGATGAGAAATGGGGTGGAAACAAAACCTACACAGCTTACG TGGACCTGGAAAAGGACTTTGCTGCTGAGGTTGTACATCCTGGAGACCTGAAGAAT TCTGTTGAAGTCGCACTGAACAAGTTGCTGGATCCAATCCGGGAAAAGTTTAATACC CCTGCCCTGAAAAAACTGGCCAGCGCTGCCTACCCAGATCCCTCAAAGCAGAAGCC AATGGCCAAAGGCCCTGCCAAGAATTCAGAACCAGAGGAGGTCATCCCATCCCGGC TGGATATCCGTGTGGGGAAAATCATCACTGTGGAGAAGCACCCAGATGCAGACAGC CTGTATGTAGAGAAGATTGACGTGGGGGAAGCTGAACCACGGACTGTGGTGAGCGG CCTGGTACAGTTCGTGCCCAAGGAGGAACTGCAGGACAGGCTGGTAGTGGTGCTGT GCAACCTGAAACCCCAGAAGATGAGAGGAGTCGAGTCCCAAGGCATGCTTCTGTGTG CTTCTATAGAAGGGATAAACCGCCAGGTTGAACCTCTGGACCCTCCGGCAGGCTCTG CTCCTGGTGAGCACGTGTTTGTGAAGGGCTATGAAAAGGGCCAACCAGATGAGGAGC TCAAGCCCAAGAAGAAAGTCTTCGAGAAGTTGCAGGCTGACTTCAAAATTTCTGAGG AGTGCATCGCACAGTGGAAGCAAACCAACTTCATGACCAAGCTGGGCTCCATTTCCT GTAAATCGCTGAAAGGGGGGAACATTAG CTAGTyrRS1^(C6) Protein/ MGDAPSPEEKLHLITRNLQEVLGEEKLKEI SEQ.ID. Human/LKERELKIYWGTATTGKPHVAYFVPMSKI NO. 38 1-197 +ADFLKAGCEVTILFADLHAYLDNMKAPWE 229-528 LLELRVSYYENVIKAMLESIGVPLEKLKFIKGTDYQLSKEYTLDVYRLSSVVTQHDSKKA GAEVVKQVEHPLLSGLLYPGLQALDEEYLKVDAQFGGIDQRKIFTFAEKESKIDLLDRK EDVKKKLKKAFCEPGNVENNGVLSFIKHVLFPLKSEFVILRDEKWGGNKTYTAYVDLE KDFAAEVVHPGDLKNSVEVALNKLLDPIREKFNTPALKKLASAAYPDPSKQKPMAKGP AKNSEPEEVIPSRLDIRVGKIITVEKHPDADSLYVEKIDVGEAEPRTVVSGLVQFVPKEEL QDRLVVVLCNLKPQKMRGVESQGMLLCASIEGINRQVEPLDPPAGSAPGEHVFVKGYE KGQPDEELKPKKKVFEKLQADFKISEECIAQWKQTNFMTKLGSISCKSLKGGNIS TyrRS1^(C6) DNA/ ATGGGGGACGCTCCCAGCCCTGAAGAGASEQ.ID. HumaN/ AACTGCACCTTATCACCCGGAACCTGCA NO. 39GGAGGTTCTGGGGGAAGAGAAGCTGAAG GAGATACTGAAGGAGCGGGAACTTAAAATTTACTGGGGAACGGCAACCACGGGCAA ACCACATGTGGCTTACTTTGTGCCCATGTCAAAGATTGCAGACTTCTTAAAGGCAGG GTGTGAGGTAACAATTCTGTTTGCGGACCTCCACGCATACCTGGATAACATGAAAGC CCCATGGGAACTTCTAGAACTCCGAGTCAGTTACTATGAGAATGTGATCAAAGCAA TGCTGGAGAGCATTGGTGTGCCCTTGGAGAAGCTCAAGTTCATCAAAGGCACTGAT TACCAGCTCAGCAAAGAGTACACACTAGATGTGTACAGACTCTCCTCCGTGGTCACA CAGCACGATTCCAAGAAGGCTGGAGCTGAGGTGGTAAAGCAGGTGGAGCACCCTTT GCTGAGTGGCCTCTTATACCCCGGACTGCAGGCTTTGGATGAAGAGTATTTAAAAGT AGATGCCCAATTTGGAGGCATTGATCAGAGAAAGATTTTCACCTTTGCAGAGAAGG AGTCCAAGATTGATCTCCTTGATCGGAAGGAGGATGTGAAGAAAAAACTGAAGAAG GCCTTCTGTGAGCCAGGAAATGTGGAGAACAATGGGGTTCTGTCCTTCATCAAGCAT GTCCTTTTTCCCCTTAAGTCCGAGTTTGTGATCCTACGAGATGAGAAATGGGGTGGA AACAAAACCTACACAGCTTACGTGGACCTGGAAAAGGACTTTGCTGCTGAGGTTGT ACATCCTGGAGACCTGAAGAATTCTGTTGAAGTCGCACTGAACAAGTTGCTGGATCC AATCCGGGAAAAGTTTAATACCCCTGCCCTGAAAAAACTGGCCAGCGCTGCCTACC CAGATCCCTCAAAGCAGAAGCCAATGGCCAAAGGCCCTGCCAAGAATTCAGAACCA GAGGAGGTCATCCCATCCCGGCTGGATATCCGTGTGGGGAAAATCATCACTGTGGA GAAGCACCCAGATGCAGACAGCCTGTATGTAGAGAAGATTGACGTGGGGGAAGCTG AACCACGGACTGTGGTGAGCGGCCTGGTACAGTTCGTGCCCAAGGAGGAACTGCAG GACAGGCTGGTAGTGGTGCTGTGCAACCTGAAACCCCAGAAGATGAGAGGAGTCGA GTCCCAAGGCATGCTTCTGTGTGCTTCTATAGAAGGGATAAACCGCCAGGTTGAACC TCTGGACCCTCCGGCAGGCTCTGCTCCTGGTGAGCACGTGTTTGTGAAGGGCTATGA AAAGGGCCAACCAGATGAGGAGCTCAAGCCCAAGAAGAAAGTCTTCGAGAAGTTGC AGGCTGACTTCAAAATTTCTGAGGAGTGCATCGCACAGTGGAAGCAAACCAACTTC ATGACCAAGCTGGGCTCCATTTCCTGTAAATCGCTGAAAGGGGGGAACATTAGCTAG TyrRS1^(C7) Protein/MGDAPSPEEKLHLITRNLQEVLGEEKLKEI SEQ.ID. Human/LKERELKIYWGTATTGKPHVAYFVPMSKI NO. 40 1-170 +ADFLKAGCEVTILFADLHAYLDNMKAPWE 229-528 LLELRVSYYENVIKAMLESIGVPLEKLKFIKGTDYQLSKEYTLDVYRLSSVVTQHDSKKA GAEVVKQVEHPLLSGLLYPGLQESKIDLLDRKEDVKKKLKKAFCEPGNVENNGVLSFIK HVLFPLKSEFVILRDEKWGGNKTYTAYVDLEKDFAAEVVHPGDLKNSVEVALNKLLDPI REKFNTPALKKLASAAYPDPSKQKPMAKGPAKNSEPEEVIPSRLDIRVGKIITVEKHPDA DSLYVEKIDVGEAEPRTVVSGLVQFVPKEELQDRLVVVLCNLKPQKMRGVESQGMLLC ASIEGINRQVEPLDPPAGSAPGEHVFVKGYEKGQPDEELKPKKKVFEKLQADFKISEECI AQWKQTNFMTKLGSISCKSLKGGNIS TyrRS1^(C7)DNA/ ATGGGGGACGCTCCCAGCCCTGAAGAGA SEQ.ID. Human/AACTGCACCTTATCACCCGGAACCTGCA NO. 41 GGAGGTTCTGGGGGAAGAGAAGCTGAAGGAGATACTGAAGGAGCGGGAACTTAAAA TTTACTGGGGAACGGCAACCACGGGCAAACCACATGTGGCTTACTTTGTGCCCATGT CAAAGATTGCAGACTTCTTAAAGGCAGGGTGTGAGGTAACAATTCTGTTTGCGGACC TCCACGCATACCTGGATAACATGAAAGCCCCATGGGAACTTCTAGAACTCCGAGTC AGTTACTATGAGAATGTGATCAAAGCAATGCTGGAGAGCATTGGTGTGCCCTTGGA GAAGCTCAAGTTCATCAAAGGCACTGATTACCAGCTCAGCAAAGAGTACACACTAG ATGTGTACAGACTCTCCTCCGTGGTCACACAGCACGATTCCAAGAAGGCTGGAGCTG AGGTGGTAAAGCAGGTGGAGCACCCTTTGCTGAGTGGCCTCTTATACCCCGGACTGC AGGAGTCCAAGATTGATCTCCTTGATCGGAAGGAGGATGTGAAGAAAAAACTGAAG AAGGCCTTCTGTGAGCCAGGAAATGTGGAGAACAATGGGGTTCTGTCCTTCATCAAG CATGTCCTTTTTCCCCTTAAGTCCGAGTTTGTGATCCTACGAGATGAGAAATGGGGTG GAAACAAAACCTACACAGCTTACGTGGACCTGGAAAAGGACTTTGCTGCTGAGGTT GTACATCCTGGAGACCTGAAGAATTCTGTTGAAGTCGCACTGAACAAGTTGCTGGAT CCAATCCGGGAAAAGTTTAATACCCCTGCCCTGAAAAAACTGGCCAGCGCTGCCTA CCCAGATCCCTCAAAGCAGAAGCCAATGGCCAAAGGCCCTGCCAAGAATTCAGAAC CAGAGGAGGTCATCCCATCCCGGCTGGATATCCGTGTGGGGAAAATCATCACTGTG GAGAAGCACCCAGATGCAGACAGCCTGTATGTAGAGAAGATTGACGTGGGGGAAGC TGAACCACGGACTGTGGTGAGCGGCCTGGTACAGTTCGTGCCCAAGGAGGAACTGC AGGACAGGCTGGTAGTGGTGCTGTGCAACCTGAAACCCCAGAAGATGAGAGGAGTC GAGTCCCAAGGCATGCTTCTGTGTGCTTCTATAGAAGGGATAAACCGCCAGGTTGAA CCTCTGGACCCTCCGGCAGGCTCTGCTCCTGGTGAGCACGTGTTTGTGAAGGGCTAT GAAAAGGGCCAACCAGATGAGGAGCTCAAGCCCAAGAAGAAAGTCTTCGAGAAGTT GCAGGCTGACTTCAAAATTTCTGAGGAGTGCATCGCACAGTGGAAGCAAACCAACT TCATGACCAAGCTGGGCTCCATTTCCTGTAAATCGCTGAAAGGGGGGAACATTAGCT AG TyrRS1^(C8) Protein/MGDAPSPEEKLHLITRNLQEVLGEEKLKEI SEQ.ID. Human/LKERELKIYWGTATTGKPHVAYFVPMSKI NO. 42 1-274 +ADFLKAGCEVTILFADLHAYLDNMKAPWE 349-528 LLELRVSYYENVIKAMLESIGVPLEKLKFIKGTDYQLSKEYTLDVYRLSSVVTQHDSKKA GAEVVKQVEHPLLSGLLYPGLQALDEEYLKVDAQFGGIDQRKIFTFAEKYLPALGYSKR VHLMNPMVPGLTGSKMSSSEEESKIDLLDRKEDVKKKLKKAFCEPGNVENNGVLSFIKH VLFPLKSEPMAKGPAKNSEPEEVIPSRLDIRVGKIITVEKHPDADSLYVEKIDVGEAEPRT VVSGLVQFVPKEELQDRLVVVLCNLKPQKMRGVESQGMLLCASIEGINRQVEPLDPPAG SAPGEHVFVKGYEKGQPDEELKPKKKVFEKLQADFKISEECIAQWKQTNFMTKLGSISC KSLKGGNIS TyrRS1^(C8) DNA/ATGGGGGACGCTCCCAGCCCTGAAGAGA SEQ.ID. Human/ AACTGCACCTTATCACCCGGAACCTGCANO. 43 GGAGGTTCTGGGGGAAGAGAAGCTGAAG GAGATACTGAAGGAGCGGGAACTTAAAATTTACTGGGGAACGGCAACCACGGGCAA ACCACATGTGGCTTACTTTGTGCCCATGTCAAAGATTGCAGACTTCTTAAAGGCAGG GTGTGAGGTAACAATTCTGTTTGCGGACCTCCACGCATACCTGGATAACATGAAAGC CCCATGGGAACTTCTAGAACTCCGAGTCAGTTACTATGAGAATGTGATCAAAGCAA TGCTGGAGAGCATTGGTGTGCCCTTGGAGAAGCTCAAGTTCATCAAAGGCACTGAT TACCAGCTCAGCAAAGAGTACACACTAGATGTGTACAGACTCTCCTCCGTGGTCACA CAGCACGATTCCAAGAAGGCTGGAGCTGAGGTGGTAAAGCAGGTGGAGCACCCTTT GCTGAGTGGCCTCTTATACCCCGGACTGCAGGCTTTGGATGAAGAGTATTTAAAAGT AGATGCCCAATTTGGAGGCATTGATCAGAGAAAGATTTTCACCTTTGCAGAGAAGT ACCTCCCTGCACTTGGCTATTCAAAACGGGTCCATCTGATGAATCCTATGGTTCCAGG ATTAACAGGCAGCAAAATGAGCTCTTCAGAAGAGGAGTCCAAGATTGATCTCCTTG ATCGGAAGGAGGATGTGAAGAAAAAACTGAAGAAGGCCTTCTGTGAGCCAGGAAAT GTGGAGAACAATGGGGTTCTGTCCTTCATCAAGCATGTCCTTTTTCCCCTTAAGTCCG AGCCAATGGCCAAAGGCCCTGCCAAGAATTCAGAACCAGAGGAGGTCATCCCATCC CGGCTGGATATCCGTGTGGGGAAAATCATCACTGTGGAGAAGCACCCAGATGCAGA CAGCCTGTATGTAGAGAAGATTGACGTGGGGGAAGCTGAACCACGGACTGTGGTGA GCGGCCTGGTACAGTTCGTGCCCAAGGAGGAACTGCAGGACAGGCTGGTAGTGGTG CTGTGCAACCTGAAACCCCAGAAGATGAGAGGAGTCGAGTCCCAAGGCATGCTTCT GTGTGCTTCTATAGAAGGGATAAACCGCCAGGTTGAACCTCTGGACCCTCCGGCAG GCTCTGCTCCTGGTGAGCACGTGTTTGTGAAGGGCTATGAAAAGGGCCAACCAGATG AGGAGCTCAAGCCCAAGAAGAAAGTCTTCGAGAAGTTGCAGGCTGACTTCAAAATT TCTGAGGAGTGCATCGCACAGTGGAAGCAAACCAACTTCATGACCAAGCTGGGCTC CATTTCCTGTAAATCGCTGAAAGGGGGG AACATTAGCTAGTyrRS1^(C9) Protein/ MTKLGSISCKSLKGGNIS SEQ.ID. Human/ NO. 44 511-528TyrRS1^(C9) DNA/ ATGACCAAGCTGGGCTCCATTTCCTGTAA SEQ.ID. Human/ATCGCTGAAAGGGGGGAACATTAGCTAG NO. 45

TABLE 2B AARS polypeptides unique splice junctions Type/Amino acid and Nucleic Acid Sequences in SEQ.ID. Name speciesthe vicinity of the unique splice junction NO. Y1-AS05 DNA/GCACCTTATCACCCGGAACCTGCAG|AGC SEQ.ID. Human/ CAATGGCCAAAGGCCCTGCCAANO. 46 Y1-AS03 DNA/ GCACCTTATCACCCGGAACCTGCAG|GTT SEQ.ID. Human/GTACATCCTGGAGACCTGAAGA NO. 47 Protein/ HLITRNLQVVHPGDLK SEQ.ID. Human/NO. 48 Y1-SV07 DNA/ GCACCTTATCACCCGGAACCTGCAG|GCT SEQ.ID. Human/TTGGATGAAGAGTATTTAAAAG NO. 49 Protein/ HLITRNLQALDEEYLK SEQ.ID. Human/NO. 50 Y1-AS02 DNA/ AAAGATTTTCACCTTTGCAGAGAAG|GAG SEQ.ID. Human/TCCAAGATTGATCTCCTTGATC NO. 51 Protein/ KIFTFAEKESKIDLLD SEQ.ID. Human/NO. 52 Y1-AS06 DNA/ TGGCCTCTTATACCCCGGACTGCAG|GAG SEQ.ID. Human/TCCAAGATTGATCTCCTTGATC NO. 53 Protein/ GLLYPGLQESKIDLLD SEQ.ID. Human/NO. 54 Y1-AS08 DNA/ CATGTCCTTTTTCCCCTTAAGTCCG|AGCC SEQ.ID. Human/AATGGCCAAAGGCCCTGCCAA NO. 55 Protein/ HVLFPLKSEPMAKGPA SEQ.ID. Human/NO. 56 Y1-AS10 DNA/ GGGGAAAATCATCACTGTGGAGAAG|AG SEQ.ID. Human/AAGGGATAAACCGCCAGGTTGAA NO. 57

TABLE 3 AARS polypeptides and nucleic acids identified by Bioinformatics 

“Protein fragments,” or the amino acid sequence of protein fragments,such as proteolytic fragments or splice variant fragments, can becharacterized, identified, or derived according to a variety oftechniques. For instance, splice variants can be identified bytechniques such as deep sequencing (see, e.g., Xing et al., RNA.14:1470-1479, 2008; and Zhang et al., Genome Research. 17:503-509,2007). As a further example, protein fragments such as proteolyticfragments can be identified in vitro, such as by incubating full-lengthor other AARS polypeptides with selected proteases, or they can beidentified endogenously (e.g., in vivo). In certain embodiments, proteinfragments such as endogenous proteolytic fragments can be generated oridentified, for instance, by recombinantly expressing full-length orother AARS polypeptides in a selected microorganism or eukaryotic cellthat has been either modified to contain one or more selected proteases,or that naturally contains one or more proteases that are capable ofacting on a selected AARS polypeptide, and isolating and characterizingthe endogenously produced protein fragments therefrom.

In certain embodiments, protein fragments such as endogenous (e.g.,naturally-occurring) proteolytic fragments can be generated oridentified, for instance, from various cellular fractions (e.g.,cytosolic, membrane, nuclear) and/or growth medium of variouscell-types, including, for example, immune cells such as monocytes,dendritic cells, macrophages (e.g., RAW 264.7 macrophages), neutrophils,eosinophils, basophils, and lymphocytes, such as B-cells and T-cells(e.g., CD4+ helper and CD8+ killer cells), including primary T-cells andT-cell lines such as Jurkat T-cells, as well as natural killer (NK)cells.

In certain embodiments, protein fragments such as endogenous proteolyticfragments, however generated, can be identified by techniques such asmass-spectrometry, or equivalent techniques. Once an in vitro orendogenously identified protein fragment has been generated oridentified, it can be mapped or sequenced, and, for example, cloned intoan expression vector for recombinant production, or producedsynthetically.

A wide variety of proteases can be used to produce, identify, derive, orcharacterize the sequence of AARS protein fragments such as proteolyticfragments. Generally, proteases are usually classified according tothree major criteria: (i) the reaction catalyzed, (ii) the chemicalnature of the catalytic site, and (iii) the evolutionary relationship,as revealed by the structure. General examples of proteases orproteinases, as classified by mechanism of catalysis, include asparticproteases, serine proteases, cysteine proteases, and metalloproteases.

Most aspartic proteases belong to the pepsin family. This familyincludes digestive enzymes, such as pepsin and chymosin, as well aslysosomal cathepsins D and processing enzymes such as renin, and certainfungal proteases (e.g., penicillopepsin, rhizopuspepsin,endothiapepsin). A second family of aspartic proteases includes viralproteinases such as the protease from the AIDS virus (HIV), also calledretropepsin.

Serine proteases include two distinct families. First, the chymotrypsinfamily, which includes the mammalian enzymes such as chymotrypsin,trypsin, elastase, and kallikrein, and second, the substilisin family,which includes the bacterial enzymes such as subtilisin. The general 3Dstructure between these two families is different, but they have thesame active site geometry, and catalysis proceeds via the samemechanism. The serine proteases exhibit different substratespecificities, differences which relate mainly to amino acidsubstitutions in the various enzyme subsites (substrate residueinteracting sites). Some serine proteases have an extended interactionsite with the substrate whereas others have a specificity that isrestricted to the P1 substrate residue.

The cysteine protease family includes the plant proteases such aspapain, actinidin, and bromelain, several mammalian lysosomalcathepsins, the cytosolic calpains (calcium-activated), as well asseveral parasitic proteases (e.g., Trypanosoma, Schistosoma). Papain isthe archetype and the best studied member of the family. Recentelucidation of the X-ray structure of the Interleukin-1-beta ConvertingEnzyme has revealed a novel type of fold for cysteine proteinases.

The metalloproteases are one of the older classes of proteases, found inbacteria, fungi, and higher organisms. They differ widely in theirsequences and their 3D structures, but the great majority of enzymescontain a zinc atom that is catalytically active. In some cases, zincmay be replaced by another metal such as cobalt or nickel without lossof proteolytic activity. Bacterial thermolysin has been wellcharacterized and its crystallographic structure indicates that zinc isbound by two histidines and one glutamic acid. Many metalloproteasescontain the sequence motif HEXXH, which provides two histidine ligandsfor the zinc. The third ligand is either a glutamic acid (thermolysin,neprilysin, alanyl aminopeptidase) or a histidine (astacin, serralysin).

Illustrative proteases include, for example, achromopeptidase,aminopeptidase, ancrod, angiotensin converting enzyme, bromelain,calpain, calpain I, calpain II, carboxypeptidase A, carboxypeptidase B,carboxypeptidase G, carboxypeptidase P, carboxypeptidase W,carboxypeptidase Y, caspase 1, caspase 2, caspase 3, caspase 4, caspase5, caspase 6, caspase 7, caspase 8, caspase 9, caspase 10, caspase 11,caspase 12, caspase 13, cathepsin B, cathepsin C, cathepsin D, cathepsinE, cathepsin G, cathepsin H, cathepsin L, chymopapain, chymase,chymotrypsin, clostripain, collagenase, complement Clr, complement Cls,complement Factor D, complement factor I, cucumisin, dipeptidylpeptidase IV, elastase (leukocyte), elastase (pancreatic),endoproteinase Arg-C, endoproteinase Asp-N, endoproteinase Glu-C,endoproteinase Lys-C, enterokinase, factor Xa, ficin, furin, granzyme A,granzyme B, HIV Protease, IGase, kallikrein tissue, leucineaminopeptidase (general), leucine aminopeptidase (cytosol), leucineaminopeptidase (microsomal), matrix metalloprotease, methionineaminopeptidase, neutrase, papain, pepsin, plasmin, prolidase, pronase E,prostate specific antigen, protease alkalophilic from Streptomycesgriseus, protease from Aspergillus, protease from Aspergillus saitoi,protease from Aspergillus sojae, protease (B. licheniformis) (alkalineor alcalase), protease from Bacillus polymyxa, protease from Bacillussp, protease from Rhizopus sp., protease S, proteasomes, proteinase fromAspergillus oryzae, proteinase 3, proteinase A, proteinase K, protein C,pyroglutamate aminopeptidase, rennin, rennin, streptokinase, subtilisin,thermolysin, thrombin, tissue plasminogen activator, trypsin, tryptaseand urokinase.

Certain embodiments relate to isolated AARS polypeptides, comprising,consisting essentially of, or consisting of amino acid sequences thathave been derived from endogenous, naturally-occurring AARS polypeptidefragments, and pharmaceutical compositions comprising said fragments,and methods of use thereof. These and related embodiments can begenerated or identified in vivo, ex vivo, and/or in vitro. In certainpreferred in vitro embodiments, AARS proteolytic fragments are generatedor identified by incubating an AARS polypeptide, such as a full-lengthAARS polypeptide, with one or more isolated human proteases, mainlythose proteases that are endogenous or natural to humans, such aselastase and others described herein and known in the art. Otherembodiments relate to isolated AARS polypeptides, comprising, consistingessentially of, or consisting of amino acid sequences that have beenderived from endogenous, naturally-occurring AARS splice variants, andpharmaceutical compositions comprising said fragments, and methods ofuse thereof. Essentially, AARS protein fragment can be isolated fromsamples that have been exposed to proteases, whether in vivo or invitro.

In certain embodiments, AARS protein fragments can be identified bytechniques such as mass-spectrometry, or equivalent techniques. Merelyby way of illustration and not limitation, in certain embodiments theproteomes from various cell types, tissues, or body fluids from avariety of physiological states (e.g., hypoxia, diet, age, disease) orfractions thereof may be separated by 1D SDS-PAGE and the gel lanes cutinto bands at fixed intervals; after which the bands may be optionallydigested with an appropriate protease, such as trypsin, to release thepeptides, which may then be analyzed by 1D reverse phase LC-MS/MS. Theresulting proteomic data may be integrated into so-called peptographs,which plot, in the left panel, sequence coverage for a given protein inthe horizontal dimension (N to C terminus, left to right) versusSDS-PAGE migration in the vertical dimension (high to low molecularweight, top to bottom). The specific peptide fragments can then besequenced or mapped. In certain embodiments, the AARS reference fragmentmay be characterized by its unique molecular weight, as compared, forexample, to the molecular weight of the corresponding full-length AARS.

As noted above, embodiments of the present invention include the AARSpolypeptides set forth in Table(s) 1-3. Also included are “variants” ofthe AARS reference polypeptides. The recitation polypeptide “variant”refers to polypeptides that are distinguished from a reference AARSpolypeptide by the addition, deletion, and/or substitution of at leastone amino acid residue, and which typically retain (e.g., mimic) ormodulate (e.g., antagonize) one or more non-canonical activities of areference AARS polypeptide.

Moreover human Tyrosyl tRNA synthetases include several hundred highlyrelated polymorphic forms, and these are known in the art to be at leastpartially functionally interchangeable. It would thus be a routinematter to select a naturally occurring variant of Tyrosyl tRNAsynthetase, including, for example the single nucleotide polymorphicforms listed in Table A to create an AARS polypeptide containing one ormore amino acid changes based on the sequence of any of the homologues,orthologs, and naturally-occurring isoforms of human as well as otherspecies of Tyrosyl tRNA synthetase.

TABLE A Human Tyrosyl tRNA synthetase SNPs Gene Bank Nucleo- Gene BankAccession tide Accession Nucleotide Number Change Number Change121908834 A/G rs1053448 C/T rs121908833 A/G rs881393 C/G rs118080042 G/Trs812518 C/G rs118041342 A/G rs796772 C/T rs116854788 C/T rs785419 C/Trs116683122 A/T rs785418 A/C rs116562938 C/G rs785417 C/T rs116535916G/T rs785416 C/T rs116510150 C/T rs785415 A/G rs116269609 A/C rs699005C/G rs116069227 C/T rs699004 A/C rs115959445 A/G rs699003 C/Trs115871830 A/C rs699002 A/T rs115869676 A/T rs3831936 -/CTAGCCCACATGCATGACTATTT rs115663825 A/G rs35490993 -/A/AAAGGGGC rs115576947 C/Trs55681162 -/AATA rs115497279 A/G rs61391327 -/A/AA rs115469699 A/Grs71006372 -/TATATATATATATA TATATATATTTT rs115286838 A/G rs71497557AT/TC rs115228430 C/G rs71497556 CAC/GTA rs115224352 A/G rs71497555ATG/GCA rs115129787 C/T rs71278770 -/TT rs114918424 C/G rs71578193GAGACTGT/TTTTTT TGAGACTGA rs114775813 A/G rs71571726 -/A rs114721539 A/Crs71571725 -/TATATA rs114691484 C/G rs71674794 -/TATATATATATATA TATATATrs114606124 A/G rs75760780 -/CTCT rs114464180 A/T rs35926575 -/Crs114401033 A/G rs35832446 -/A rs114400708 A/G rs35746182 C/Trs114366281 C/G rs35718370 -/A rs114349735 A/C rs35712477 G/Trs114342901 A/G rs35670866 -/T rs114223825 C/T rs35504526 -/Grs113890790 A/C rs35498629 -/G rs113889404 A/G rs35468803 -/Crs113783279 A/G rs35431920 -/T rs113773781 C/T rs35929714 -/CTrs113748028 C/T rs6677618 C/T rs113726588 C/T rs6673913 A/T rs113680203A/G rs6425801 C/T rs113641360 A/G rs5773386 -/T rs113552541 A/Grs4951787 A/G rs113519565 -/T rs4951786 C/G rs113502268 A/G rs4951785C/G rs113379352 C/T rs4951784 C/G rs113358843 C/G rs4951783 C/Trs113311797 G/T rs4454511 G/T rs113236259 A/G rs4451524 A/T rs113087774C/G rs4451523 A/T rs112960756 C/T rs4422972 G/T rs112854851 C/Trs3965414 A/T rs112812557 A/G rs3894376 C/T rs112764558 A/G rs3894375C/T rs112714303 A/G rs3845499 A/G rs112700864 G/T rs3737252 C/Trs112699523 A/C rs3737251 C/G rs112694417 G/T rs2786593 C/T rs112637129-/T rs2786546 A/G rs112629040 A/G rs2449459 G/T rs112540487 C/Trs2335410 C/T rs112443525 A/C rs2282294 A/T rs112375264 A/G rs2282293A/G rs112278462 C/T rs2128600 G/T rs112271329 C/T rs1765231 A/Grs112104823 C/T rs1765230 C/T rs112044368 C/T rs1765226 A/G rs112037794A/G rs1765223 G/T rs112036210 C/T rs1764914 G/T rs111980731 A/Trs1764903 A/G rs111777976 A/G rs1681001 A/G rs111752899 C/G rs1143194C/G rs111690225 C/T rs1085230 C/G rs111651315 C/G rs1085229 C/Trs111568458 C/T rs1085228 A/C rs111541973 G/T rs1085227 C/T rs111454813C/G rs1053482 A/G rs111411658 -/A rs12126642 A/G rs111331078 A/Grs12045765 A/C rs111303111 A/G rs12042173 C/G rs111244678 A/G rs12040000C/T rs80332217 A/G rs12036018 C/T rs80253625 A/T rs12035128 C/Trs80154641 C/T rs12032397 A/G rs79815772 A/C rs12032306 C/T rs79791410A/G rs12027991 A/G rs79738434 C/G rs12027177 A/G rs79710362 A/Grs11809784 A/C rs79622512 C/T rs11806080 A/C rs79585772 A/C rs11799668C/T rs79302557 C/T rs11587620 C/G rs79272803 A/G rs11583112 A/Grs79255128 A/T rs11581652 C/T rs79240354 -/T rs11544327 A/C rs79239661C/G rs11544326 G/T rs78950256 C/T rs11544325 C/T rs78903576 A/Crs11544324 C/T rs78850379 A/G rs11544323 C/T rs78598973 A/C rs11544322C/T rs78433384 A/G rs11433443 -/A rs78320277 G/T rs11381151 -/Ars78319035 A/C rs11344284 -/T rs78090292 A/T rs11318209 -/T rs77933298A/T rs10914621 C/T rs77922346 C/T rs10914619 A/G rs77573475 C/Trs10914617 A/T rs77572572 A/G rs10914614 A/G rs77564909 C/T rs10914613C/T rs77508723 A/G rs10914611 G/T rs77395170 C/G rs10798918 C/Trs77372558 A/G rs10753265 A/G rs77240034 A/G rs9787263 A/C rs77174196A/T rs7548570 A/G rs77116473 A/C rs7539432 C/G rs77033253 -/AT rs7411174A/G rs76991974 A/C rs6686315 A/G rs76862302 C/T rs35381223 C/Grs76657858 A/C rs35287970 -/G rs76611863 G/T rs35270871 -/T rs76610248C/G rs35204694 -/C rs76497653 A/C rs34834736 -/T rs76460696 A/Crs34800768 -/T rs76435574 C/T rs34768899 -/T rs76428484 A/C rs34704129-/C rs76313514 C/T rs34700143 A/G rs76239948 C/T rs34619886 C/Trs76213478 G/T rs34553868 G/T rs76172167 C/T rs34533197 C/T rs76154286A/G rs34506343 -/A rs76066944 A/C rs34460415 C/T rs75863818 A/Trs34458777 -/C rs75658271 A/G rs34412390 -/T rs75629042 A/C rs34366587C/T rs75556988 A/G rs34305494 -/T rs75506593 G/T rs34297917 -/Trs75499764 A/C rs34270752 -/T rs75457356 C/T rs34240771 A/T rs75420675A/G rs34213904 -/T rs75382318 A/T rs34204340 -/T rs74852498 -/CAArs34189200 -/T rs74403587 A/C rs34134602 -/G rs74319398 C/T rs34054038-/C rs74067514 C/T rs34046956 -/A rs73618880 A/G rs28390180 A/Grs72880571 C/T rs17510718 C/T rs72880567 A/G rs16866009 C/T rs72880562C/T rs16835176 A/G rs72880555 G/T rs12760548 G/T rs72880553 A/Grs12759668 A/G rs72880540 A/G rs12729898 G/T rs72874656 A/G rs12724469G/T rs72654022 A/G rs12564655 C/T rs72654020 A/C rs12406682 C/Trs72654016 A/G rs12136633 C/G rs72510634 -/CT rs12133278 A/G rs72418507-/AA rs59754854 C/T rs72391325 -/T rs59697437 A/T rs72315501 -/AATArs59686477 C/T rs72147372 -/T rs59542013 -/A rs72089012 -/AA rs59347601C/T rs72050208 -/T rs59076866 A/C rs71910487 -/T rs58994767 -/Trs71772495 -/T rs58957786 C/T rs71665854 -/A rs58938776 C/T rs71646400C/T rs58905217 G/T rs71646399 A/G rs58828752 C/T rs71646397 C/Grs58621825 -/T rs71516333 C/T rs58440351 A/T rs71516332 C/G rs57928583C/T rs71516331 C/G rs57734982 A/T rs71006371 -/A rs57578136 -/Trs71006370 -/T rs57215825 C/G rs68183151 A/C rs56697788 -/AA rs68073354-/T rs56064318 C/T rs67839900 -/A rs56023272 A/G rs67504155 -/Ars41265865 A/G rs67210611 -/T rs41265863 G/T rs67020459 -/TC rs41265861A/G rs66590952 -/CT rs41265859 A/G rs61800867 A/G rs41265857 C/Trs61800866 C/T rs36092637 A/T rs61798853 C/G rs36061373 -/C rs61798852A/G rs36056058 C/T rs61798851 C/T rs61798849 C/T rs61798846 A/Grs61798845 A/G rs61737106 A/T rs61057925 A/G rs60958989 C/T rs60802151C/T rs60704395 -/T rs60269206 A/G rs60246059 C/G

In certain embodiments, a polypeptide variant is distinguished from areference polypeptide by one or more substitutions, which may beconservative or non-conservative, as described herein and known in theart. In certain embodiments, the polypeptide variant comprisesconservative substitutions and, in this regard, it is well understood inthe art that some amino acids may be changed to others with broadlysimilar properties without changing the nature of the activity of thepolypeptide.

In certain embodiments, a variant polypeptide includes an amino acidsequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or more sequence identity orsimilarity to a corresponding sequence of an AARS reference polypeptide,as described herein, and substantially retains the non-canonicalactivity of that reference polypeptide. Also included are sequencesdiffering from the reference AARS sequences by the addition, deletion,or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140,150 or more amino acids but which retain the properties of the referenceAARS polypeptide. In certain embodiments, the amino acid additions ordeletions occur at the C-terminal end and/or the N-terminal end of theAARS reference polypeptide. In certain embodiments, the amino acidadditions include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 30, 40, 50 or more wild-type residues (i.e., from thecorresponding full-length AARS polypeptide) that are proximal to theC-terminal end and/or the N-terminal end of the AARS referencepolypeptide.

In certain embodiments, variant polypeptides differ from thecorresponding AARS reference sequences by at least 1% but less than 20%,15%, 10% or 5% of the residues. (If this comparison requires alignment,the sequences should be aligned for maximum similarity. “Looped” outsequences from deletions or insertions, or mismatches, are considereddifferences.) The differences are, suitably, differences or changes at anon-essential residue or a conservative substitution. In certainembodiments, the molecular weight of a variant AARS polypeptide differsfrom that of the AARS reference polypeptide by about 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,or more.

Also included are biologically active “fragments” of the AARS referencepolypeptides, i.e., biologically active fragments of the AARS proteinfragments. Representative biologically active fragments generallyparticipate in an interaction, e.g., an intramolecular or aninter-molecular interaction. An inter-molecular interaction can be aspecific binding interaction or an enzymatic interaction. Aninter-molecular interaction can be between an AARS polypeptide and acellular binding partner, such as a cellular receptor or other hostmolecule that participates in the non-canonical activity of the AARSpolypeptide. In some embodiments, AARS proteins, variants, andbiologically active fragments thereof, bind to one or more cellularbinding partners with an affinity of at least about 0.01, 0.05, 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 40, or 50 nM. The binding affinity of an AARS protein fragmentfor a selected cellular binding partner, particularly a binding partnerthat participates in a non-canonical activity, is typically strongerthan that of the AARS protein fragment's corresponding full-length AARSpolypeptide, by at least about 1.5×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×,6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×, 30×, 40×, 50×, 60×, 70×, 80×, 90×,100×, 200×, 300×, 400×, 500×, 600×, 700×, 800×, 900×, 1000× or more(including all integers in between). The binding affinity of an AARSprotein fragment for a binding partner that participates in at least onecanonical activity of an AARS is typically weaker than that of the AARSprotein fragment's corresponding full-length AARS polypeptide, by atleast about 1.5×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×,15×, 20×, 25×, 30×, 40×, 50×, 60×, 70×, 80×, 90×, 100×, 200×, 300×,400×, 500×, 600×, 700×, 800×, 900×, 1000× or more.

Typically, biologically active fragments comprise a domain or motif withat least one activity of an AARS reference polypeptide and may includeone or more (and in some cases all) of the various active domains, andinclude fragments having a non-canonical activity. In some cases,biologically active fragments of an AARS polypeptide have a biologicalactivity that is unique to the particular, truncated fragment, such thatthe full-length AARS polypeptide may not have that activity. In certaincases, the biological activity may be revealed by separating thebiologically active AARS polypeptide fragment from the other full-lengthAARS polypeptide sequences, or by altering certain residues of thefull-length AARS wild-type polypeptide sequence to unmask thebiologically active domains.

A biologically active fragment of an AARS reference polypeptide can be apolypeptide fragment which is, for example, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50,55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160,170, 180, 190, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400,450, 500, 550, 600, 650, 700, 750 or more contiguous or non-contiguousamino acids, including all integers (e.g., 101, 102, 103) and ranges(e.g., 50-100, 50-150, 50-200) in between, of the amino acid sequencesset forth in any one of the AARS reference polypeptides describedherein, but typically exclude the full-length AARS. In certainembodiments, a biologically active fragment comprises a non-canonicalactivity-related sequence, domain, or motif. In certain embodiments, theC-terminal or N-terminal region of any AARS reference polypeptide may betruncated by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35,40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, or 700 or moreamino acids, or by about 10-50, 20-50, 50-100, 100-150, 150-200,200-250, 250-300, 300-350, 350-400, 400-450, 450-500, 500-550, 550-600,600-650, 650-700 or more amino acids, including all integers and rangesin between (e.g., 101, 102, 103, 104, 105), so long as the truncatedAARS polypeptide retains the non-canonical activity of the referencepolypeptide. Typically, the biologically-active fragment has no lessthan about 1%, about 5%, about 10%, about 25%, or about 50% of anactivity of the biologically-active (i.e., non-canonical activity) AARSreference polypeptide from which it is derived. Exemplary methods formeasuring such non-canonical activities are described in the Examples.

As noted above, an AARS polypeptide may be altered in various waysincluding amino acid substitutions, deletions, truncations, andinsertions. Methods for such manipulations are generally known in theart. For example, amino acid sequence variants of an AARS referencepolypeptide can be prepared by mutations in the DNA. Methods formutagenesis and nucleotide sequence alterations are well known in theart. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82:488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367-382), U.S.Pat. No. 4,873,192, Watson, J. D. et al., (“Molecular Biology of theGene”, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif, 1987) andthe references cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al., (1978) Atlas ofProtein Sequence and Structure (Natl. Biomed. Res. Found., Washington,D.C.).

Similarly it is within the skill in the art to address and/or mitigateimmunogenicity concerns if they arise using an AARS polypeptide, e.g.,by the use of automated computer recognition programs to identifypotential T cell epitopes, and directed evolution approaches to identifyless immunogenic forms.

Methods for screening gene products of combinatorial libraries made bypoint mutations or truncation, and for screening cDNA libraries for geneproducts having a selected property are known in the art. Such methodsare adaptable for rapid screening of the gene libraries generated bycombinatorial mutagenesis of AARS polypeptides. Recursive ensemblemutagenesis (REM), a technique which enhances the frequency offunctional mutants in the libraries, can be used in combination with thescreening assays to identify AARS polypeptide variants (Arkin andYourvan (1992) Proc. Natl. Acad. Sci. USA 89: 7811-7815; Delgrave etal., (1993) Protein Engineering, 6: 327-331). Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be desirable as discussed in more detail below.

Biologically active truncated and/or variant AARS polypeptides maycontain conservative amino acid substitutions at various locations alongtheir sequence, as compared to a reference AARS amino acid residue.Additionally, naturally occurring variants of AARS proteins have beensequenced, and are known in the art to be at least partiallyfunctionally interchangeable. It would thus be a routine matter toselect an amino acid position to introduce a conservative, or nonconservative mutation into an AARS polypeptide based on naturallyoccurring sequence variation among the known AARS protein homologues,orthologs, and naturally-occurring isoforms of human as well as otherspecies of an AARS protein.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, which can be generally sub-classified asfollows:

Acidic: The residue has a negative charge due to loss of H ion atphysiological pH and the residue is attracted by aqueous solution so asto seek the surface positions in the conformation of a peptide in whichit is contained when the peptide is in aqueous medium at physiologicalpH. Amino acids having an acidic side chain include glutamic acid andaspartic acid.

Basic: The residue has a positive charge due to association with H ionat physiological pH or within one or two pH units thereof (e.g.,histidine) and the residue is attracted by aqueous solution so as toseek the surface positions in the conformation of a peptide in which itis contained when the peptide is in aqueous medium at physiological pH.Amino acids having a basic side chain include arginine, lysine andhistidine.

Charged: The residues are charged at physiological pH and, therefore,include amino acids having acidic or basic side chains (i.e., glutamicacid, aspartic acid, arginine, lysine and histidine).

Hydrophobic: The residues are not charged at physiological pH and theresidue is repelled by aqueous solution so as to seek the innerpositions in the conformation of a peptide in which it is contained whenthe peptide is in aqueous medium. Amino acids having a hydrophobic sidechain include tyrosine, valine, isoleucine, leucine, methionine,phenylalanine and tryptophan.

Neutral/polar: The residues are not charged at physiological pH, but theresidue is not sufficiently repelled by aqueous solutions so that itwould seek inner positions in the conformation of a peptide in which itis contained when the peptide is in aqueous medium. Amino acids having aneutral/polar side chain include asparagine, glutamine, cysteine,histidine, serine and threonine.

This description also characterizes certain amino acids as “small” sincetheir side chains are not sufficiently large, even if polar groups arelacking, to confer hydrophobicity. With the exception of proline,“small” amino acids are those with four carbons or less when at leastone polar group is on the side chain and three carbons or less when not.Amino acids having a small side chain include glycine, serine, alanineand threonine. The gene-encoded secondary amino acid proline is aspecial case due to its known effects on the secondary conformation ofpeptide chains. The structure of proline differs from all the othernaturally-occurring amino acids in that its side chain is bonded to thenitrogen of the α-amino group, as well as the α-carbon. Several aminoacid similarity matrices are known in the art (see e.g., PAM120 matrixand PAM250 matrix as disclosed for example by Dayhoff et al., 1978, Amodel of evolutionary change in proteins). Matrices for determiningdistance relationships In M. O. Dayhoff, (ed.), Atlas of proteinsequence and structure, Vol. 5, pp. 345-358, National BiomedicalResearch Foundation, Washington D.C.; and by Gonnet et al., (Science,256: 14430-1445, 1992), however, include proline in the same group asglycine, serine, alanine and threonine. Accordingly, for the purposes ofthe present invention, proline is classified as a “small” amino acid.

The degree of attraction or repulsion required for classification aspolar or nonpolar is arbitrary and, therefore, amino acids specificallycontemplated by the invention have been classified as one or the other.Most amino acids not specifically named can be classified on the basisof known behavior.

Amino acid residues can be further sub-classified as cyclic ornon-cyclic, and aromatic or non-aromatic, self-explanatoryclassifications with respect to the side-chain substituent groups of theresidues, and as small or large. The residue is considered small if itcontains a total of four carbon atoms or less, inclusive of the carboxylcarbon, provided an additional polar substituent is present; three orless if not. Small residues are, of course, always non-aromatic.Dependent on their structural properties, amino acid residues may fallin two or more classes. For the naturally-occurring protein amino acids,sub-classification according to this scheme is presented in Table B.

TABLE B Amino acid sub-classification Sub-classes Amino acids AcidicAspartic acid, Glutamic acid Basic Noncyclic: Arginine, Lysine; Cyclic:Histidine Charged Aspartic acid, Glutamic acid, Arginine, Lysine,Histidine Small Glycine, Serine, Alanine, Threonine, ProlinePolar/neutral Asparagine, Histidine, Glutamine, Cysteine, Serine,Threonine Polar/large Asparagine, Glutamine Hydrophobic Tyrosine,Valine, Isoleucine, Leucine, Methionine, Phenylalanine, TryptophanAromatic Tryptophan, Tyrosine, Phenylalanine Residues that Glycine andProline influence chain orientation

Conservative amino acid substitution also includes groupings based onside chains. For example, a group of amino acids having aliphatic sidechains is glycine, alanine, valine, leucine, and isoleucine; a group ofamino acids having aliphatic-hydroxyl side chains is serine andthreonine; a group of amino acids having amide-containing side chains isasparagine and glutamine; a group of amino acids having aromatic sidechains is phenylalanine, tyrosine, and tryptophan; a group of aminoacids having basic side chains is lysine, arginine, and histidine; and agroup of amino acids having sulfur-containing side chains is cysteineand methionine. For example, it is reasonable to expect that replacementof a leucine with an isoleucine or valine, an aspartate with aglutamate, a threonine with a serine, or a similar replacement of anamino acid with a structurally related amino acid will not have a majoreffect on the properties of the resulting variant polypeptide. Whetheran amino acid change results in a functional truncated and/or variantAARS polypeptide can readily be determined by assaying its non-canonicalactivity, as described herein. Conservative substitutions are shown inTable C under the heading of exemplary substitutions. Amino acidsubstitutions falling within the scope of the invention, are, ingeneral, accomplished by selecting substitutions that do not differsignificantly in their effect on maintaining (a) the structure of thepeptide backbone in the area of the substitution, (b) the charge orhydrophobicity of the molecule at the target site, (c) the bulk of theside chain, or (d) the biological function. After the substitutions areintroduced, the variants are screened for biological activity.

TABLE C Exemplary Amino Acid Substitutions Original Exemplary PreferredResidue Substitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln,Asn Lys Asn Gln, His, Lys, Arg Gln Asp Glu Glu Cys Ser Ser Gln Asn, His,Lys, Asn Glu Asp, Lys Asp Gly Pro Pro His Asn, Gln, Lys, Arg Arg IleLeu, Val, Met, Ala, Phe, Norleu Leu Leu Norleu, Ile, Val, Met, Ala, PheIle Lys Arg, Gln, Asn Arg Met Leu, Ile, Phe Leu Phe Leu, Val, Ile, AlaLeu Pro Gly Gly Ser Thr Thr Thr Ser Ser Trp Tyr Tyr Tyr Trp, Phe, Thr,Ser Phe Val Ile, Leu, Met, Phe, Ala, Norleu Leu

Alternatively, similar amino acids for making conservative substitutionscan be grouped into three categories based on the identity of the sidechains. The first group includes glutamic acid, aspartic acid, arginine,lysine, histidine, which all have charged side chains; the second groupincludes glycine, serine, threonine, cysteine, tyrosine, glutamine,asparagine; and the third group includes leucine, isoleucine, valine,alanine, proline, phenylalanine, tryptophan, methionine, as described inZubay, G., Biochemistry, third edition, Wm.C. Brown Publishers (1993).

Thus, a predicted non-essential amino acid residue in a truncated and/orvariant AARS polypeptide is typically replaced with another amino acidresidue from the same side chain family. Alternatively, mutations can beintroduced randomly along all or part of an AARS coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor an activity of the parent polypeptide to identify mutants whichretain that activity. Following mutagenesis of the coding sequences, theencoded peptide can be expressed recombinantly and the activity of thepeptide can be determined. A “non-essential” amino acid residue is aresidue that can be altered from the reference sequence of an embodimentpolypeptide without abolishing or substantially altering one or more ofits activities. Suitably, the alteration does not substantially abolishone of these activities, for example, the activity is at least 20%, 40%,60%, 70% or 80% 100%, 500%, 1000% or more of the reference AARSsequence. An “essential” amino acid residue is a residue that, whenaltered from the reference sequence of an AARS polypeptide, results inabolition of an activity of the parent molecule such that less than 20%of the reference activity is present. For example, such essential aminoacid residues include those that are conserved in AARS polypeptidesacross different species, including those sequences that are conservedin the active binding site(s) or motif(s) of AARS polypeptides fromvarious sources.

In general, polypeptides and fusion polypeptides (as well as theirencoding polynucleotides) are isolated. An “isolated” polypeptide orpolynucleotide is one that is removed from its original environment. Forexample, a naturally-occurring protein is isolated if it is separatedfrom some or all of the coexisting materials in the natural system.Preferably, such polypeptides are at least about 90% pure, morepreferably at least about 95% pure and most preferably at least about99% pure. A polynucleotide is considered to be isolated if, for example,it is cloned into a vector that is not a part of the naturalenvironment.

Certain embodiments also encompass dimers of AARS polypeptides. Dimersmay include, for example, homodimers between two identical AARSpolypeptides, heterodimers between two different AARS polypeptides(e.g., a full-length YRS polypeptide and a truncated YRS polypeptide; atruncated YRS polypeptide and a truncated WRS polypeptide), and/orheterodimers between an AARS polypeptide and a heterologous polypeptide.Certain heterodimers, such as those between an AARS polypeptide and aheterologous polypeptide, may be bi-functional, as described herein.

Also included are monomers of AARS polypeptides, including isolated AARSpolypeptides monomers that do not substantially dimerize with a secondAARS polypeptide, whether due to one or more substitutions, truncations,deletions, additions, chemical modifications, or a combination of thesealterations. In certain embodiments, monomeric AARS polypeptides possessbiological activities, including non-canonical activities, which are notpossessed by dimeric or multimeric AARS polypeptide complexes.

Certain embodiments of the present invention also contemplate the use ofmodified AARS polypeptides, including modifications that improved thedesired characteristics of an AARS polypeptide, as described herein.Modifications of AARS polypeptides of the invention include chemicaland/or enzymatic derivatizations at one or more constituent amino acid,including side chain modifications, backbone modifications, and N- andC-terminal modifications including acetylation, hydroxylation,methylation, amidation, and the attachment of carbohydrate or lipidmoieties, cofactors, and the like. Exemplary modifications also includepegylation of an AARS polypeptide (see, e.g., Veronese and Harris,Advanced Drug Delivery Reviews 54: 453-456, 2002; and Pasut et al.,Expert Opinion. Ther. Patents 14(6) 859-894 2004, both hereinincorporated by reference).

PEG is a well-known polymer having the properties of solubility in waterand in many organic solvents, lack of toxicity, and lack ofimmunogenicity. It is also clear, colorless, odorless, and chemicallystable. For these reasons and others, PEG has been selected as thepreferred polymer for attachment, but it has been employed solely forpurposes of illustration and not limitation. Similar products may beobtained with other water-soluble polymers, including withoutlimitation; polyvinyl alcohol, other poly(alkylene oxides) such aspoly(propylene glycol) and the like, poly(oxyethylated polyols) such aspoly(oxyethylated glycerol) and the like, carboxymethylcellulose,dextran, polyvinyl alcohol, polyvinyl purrolidone, poly-1,3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride, and polyaminoacids. Oneskilled in the art will be able to select the desired polymer based onthe desired dosage, circulation time, resistance to proteolysis, andother considerations.

In particular a wide variety of PEG derivatives are both available andsuitable for use in the preparation of PEG-conjugates. For example, NOFCorp.'s PEG reagents sold under the trademark SUNBRIGHT® Series providesnumerous PEG derivatives, including methoxypolyethylene glycols andactivated PEG derivatives such as methoxy-PEG amines, maleimides,N-hydroxysuccinimide esters, and carboxylic acids, for coupling byvarious methods to the N-terminal, C-terminal or any internal amino acidof the AARS polypeptide. Nektar Therapeutics' Advanced PEGylationtechnology also offers diverse PEG-coupling technologies to potentiallyimprove the safety and efficacy of an AARS polypeptide basedtherapeutic.

A search of patents, published patent applications, and relatedpublications will also provide those skilled in the art reading thisdisclosure with significant possible PEG-coupling technologies andPEG-derivatives. For example, U.S. Pat. Nos. 6,436,386; 5,932,462;5,900,461; 5,824,784; and 4,904,584; the contents of which areincorporated by reference in their entirety, describe such technologiesand derivatives, and methods for their manufacture.

In certain aspects, chemoselective ligation technology may be utilizedto modify AARS polypeptides of the invention, such as by attachingpolymers in a site-specific and controlled manner. Such technologytypically relies on the incorporation of chemoselective anchors into theprotein backbone by either chemical, or recombinant means, andsubsequent modification with a polymer carrying a complementary linker.As a result, the assembly process and the covalent structure of theresulting protein—polymer conjugate may be controlled, enabling therational optimization of drug properties, such as efficacy andpharmacokinetic properties (see, e.g., Kochendoerfer, Current Opinion inChemical Biology 9:555-560, 2005).

In other embodiments, fusion proteins of AARS polypeptide to otherproteins are also included, and these fusion proteins may increase theAARS polypeptide's biological activity, secretion, targeting, biologicallife, ability to penetrate cellular membranes, or the blood brainbarrier, or pharmacokinetic properties. Examples of fusion proteins thatimprove pharmacokinetic properties (“PK modifiers”) include withoutlimitation, fusions to human albumin (Osborn et al.: Eur. J. Pharmacol.456(1-3): 149-158, (2002)), antibody Fc domains, poly Glu or poly Aspsequences, and transferrin. Additionally, fusion with conformationallydisordered polypeptide sequences composed of the amino acids Pro, Ala,and Ser (‘PASylation’) or hydroxyethyl starch (sold under the trademarkHESYLATION®) provides a simple way to increase the hydrodynamic volumeof the AARS polypeptide. This additional extension adopts a bulky randomstructure, which significantly increases the size of the resultingfusion protein. By this means the typically rapid clearance of smallerAARS polypeptides via kidney filtration is retarded by several orders ofmagnitude. Additionally use of Ig G fusion proteins has also been shownto enable some fusion protein proteins to penetrate the blood brainbarrier (Fu et al., (2010) Brain Res. 1352:208-13).

Examples of fusion proteins that improve penetration across cellularmembranes include fusions to membrane translocating sequences. In thiscontext, the term “membrane translocating sequences” refers to naturallyoccurring and synthetic amino acid sequences that are capable ofmembrane translocation across a cellular membrane. Representativemembrane translocating sequences include those based on the naturallyoccurring membrane translocating sequences derived from the Tat protein,and homeotic transcription protein Antennapedia, as well as syntheticmembrane translocating sequences based in whole or part on poly Arginineand Lysine resides. Representative membrane translocating sequencesinclude for example those disclosed in the following patents, U.S. Pat.No. 5,652,122; U.S. Pat. No. 5,670,617; U.S. Pat. No. 5,674,980; U.S.Pat. No. 5,747,641; U.S. Pat. No. 5,804,604; U.S. Pat. No. 6,316,003;U.S. Pat. No. 7,585,834; U.S. Pat. No. 7,312,244; U.S. Pat. No.7,279,502; U.S. Pat. No. 7,229,961; U.S. Pat. No. 7,169,814; U.S. Pat.No. 7,453,011; U.S. Pat. No. 7,235,695; U.S. Pat. No. 6,982,351; U.S.Pat. No. 6,605,115; U.S. Pat. No. 7,306,784; U.S. Pat. No. 7,306,783;U.S. Pat. No. 6,589,503; U.S. Pat. No. 6,348,185; U.S. Pat. No.6,881,825; U.S. Pat. No. 7,431,915; WO0074701A2; WO2007111993A2;WO2007106554A2; WO02069930A1; WO03049772A2; WO03106491A2; andWO2008063113A1.

It will be appreciated that a flexible molecular linker (or spacer)optionally may be interposed between, and covalently join, the AARSpolypeptide and any of the fusion proteins disclosed herein.

Additionally in some embodiments, the AARS polypeptide can includesynthetic, or naturally occurring secretion signal sequences, derivedfrom other well characterized secreted proteins. In some embodimentssuch proteins, may be processed by proteolytic cleavage to form the AARSpolypeptide in situ. Such fusions proteins include for example fusionsof AARS polypeptide to ubiquitin to provide a new N-terminal amino acid,or the use of a secretion signal to mediate high level secretion of theAARS polypeptide into the extracellular medium, or N, or C-terminalepitope tags to improve purification or detection.

The AARS polypeptides described herein may be prepared by any suitableprocedure known to those of skill in the art, such as by recombinanttechniques. In addition to recombinant production methods, polypeptidesof the invention may be produced by direct peptide synthesis usingsolid-phase techniques (Merrifield, J. Am. Chem. Soc. 85:2149-2154(1963)). Protein synthesis may be performed using manual techniques orby automation. Automated synthesis may be achieved, for example, usingApplied Biosystems 431A Peptide Synthesizer (Perkin Elmer).Alternatively, various fragments may be chemically synthesizedseparately and combined using chemical methods to produce the desiredmolecule.

IV. AARS POLYNUCLEOTIDES

Embodiments of the present invention include polynucleotides that encodeone or more newly identified protein fragments of an aminoacyl-tRNAsynthetase (AARS), in addition to complements, variants, and fragmentsthereof. In certain embodiments, an AARS polynucleotide encodes all or aportion of the AARS polypeptide reference sequence(s) as set forth inTable(s) 1-3, which represent splice variants, proteolytic fragments, orother type of fragments of tyrosyl tRNA synthetase. Certain embodimentsinclude polynucleotides, encoding polypeptides or proteins that comprisethe sequence of one or more splice junctions of those splice variants,in addition to complements, variants, and fragments thereof. In certainembodiments, typically due to the singular nature of a selected AARSsplice variant, which combines exons in a new or exceptional way, theAARS polynucleotide references sequences comprise a unique orexceptional splice junction. Certain embodiments exclude a correspondingfull-length AARS polynucleotide.

Also included within the AARS polynucleotides of the present inventionare primers, probes, antisense oligonucleotides, and RNA interferenceagents that comprise all or a portion of these referencepolynucleotides, which are complementary to all or a portion of thesereference polynucleotides, or which specifically hybridize to thesereference polynucleotides, as described herein.

The term “polynucleotide” or “nucleic acid” as used herein designatesmRNA, RNA, cRNA, cDNA or DNA. The term typically refers to polymericform of nucleotides of at least 10 bases in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide. The term includes single and double stranded forms of DNA.The terms “DNA” and “polynucleotide” and “nucleic acid” refer to a DNAmolecule that has been isolated free of total genomic DNA of aparticular species. Therefore, an isolated DNA segment encoding apolypeptide refers to a DNA segment that contains one or more codingsequences yet is substantially isolated away from, or purified freefrom, total genomic DNA of the species from which the DNA segment isobtained. Also included are non-coding polynucleotides (e.g., primers,probes, oligonucleotides), which do not encode an AARS polypeptide.Included within the terms “DNA segment” and “polynucleotide” are DNAsegments and smaller fragments of such segments, and also recombinantvectors, including, for example, plasmids, cosmids, phagemids, phage,viruses, and the like.

Additional coding or non-coding sequences may, but need not, be presentwithin a polynucleotide of the present invention, and a polynucleotidemay, but need not, be linked to other molecules and/or supportmaterials. Hence, the polynucleotides of the present invention,regardless of the length of the coding sequence itself, may be combinedwith other DNA sequences, such as promoters, polyadenylation signals,additional restriction enzyme sites, multiple cloning sites, othercoding segments, and the like, such that their overall length may varyconsiderably.

It is therefore contemplated that a polynucleotide fragment of almostany length may be employed; with the total length preferably beinglimited by the ease of preparation and use in the intended recombinantDNA protocol. Included are polynucleotides of about 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 41, 43, 44, 45, 46, 47, 48, 49, 50,60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200,220, 240, 260, 270, 280, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700,1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900,3000 or more (including all integers in between) bases in length,including any portion or fragment (e.g., greater than about 6, 7, 8, 9,or 10 nucleotides in length) of an AARS reference polynucleotide (e.g.,base number X-Y, in which X is about 1-3000 or more and Y is about10-3000 or more), or its complement.

Embodiments of the present invention also include “variants” of the AARSreference polynucleotide sequences. Polynucleotide “variants” maycontain one or more substitutions, additions, deletions and/orinsertions in relation to a reference polynucleotide. Generally,variants of an AARS reference polynucleotide sequence may have at leastabout 30%, 40% 50%, 55%, 60%, 65%, 70%, generally at least about 75%,80%, 85%, desirably about 90% to 95% or more, and more suitably about98% or more sequence identity to that particular nucleotide sequence asdetermined by sequence alignment programs described elsewhere hereinusing default parameters. In certain embodiments, variants may differfrom a reference sequence by about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 41, 43, 44, 45, 46, 47,48, 49, 50, 60, 70, 80, 90, 100 (including all integers in between) ormore bases. In certain embodiments, such as when the polynucleotidevariant encodes an AARS polypeptide having a non-canonical activity, thedesired activity of the encoded AARS polypeptide is not substantiallydiminished relative to the unmodified polypeptide. The effect on theactivity of the encoded polypeptide may generally be assessed asdescribed herein.

Certain embodiments include polynucleotides that hybridize to areference AARS polynucleotide sequence, or to their complements, understringency conditions described below. As used herein, the term“hybridizes under low stringency, medium stringency, high stringency, orvery high stringency conditions” describes conditions for hybridizationand washing. Guidance for performing hybridization reactions can befound in Ausubel et al., (1998, supra), Sections 6.3.1-6.3.6. Aqueousand non-aqueous methods are described in that reference and either canbe used.

Reference herein to low stringency conditions include and encompass fromat least about 1% v/v to at least about 15% v/v formamide and from atleast about 1 M to at least about 2 M salt for hybridization at 42° C.,and at least about 1 M to at least about 2 M salt for washing at 42° C.Low stringency conditions also may include 1% Bovine Serum Albumin(BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65°C., and (i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄(pH 7.2), 5% SDS for washing at room temperature. One embodiment of lowstringency conditions includes hybridization in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by two washes in0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes canbe increased to 55° C. for low stringency conditions).

Medium stringency conditions include and encompass from at least about16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9 M salt for hybridization at 42° C., and at leastabout 0.1 M to at least about 0.2 M salt for washing at 55° C. Mediumstringency conditions also may include 1% Bovine Serum Albumin (BSA), 1mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and(i) 2×SSC, 0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2),5% SDS for washing at 60-65° C. One embodiment of medium stringencyconditions includes hybridizing in 6×SSC at about 45° C., followed byone or more washes in 0.2×SSC, 0.1% SDS at 60° C. High stringencyconditions include and encompass from at least about 31% v/v to at leastabout 50% v/v formamide and from about 0.01 M to about 0.15 M salt forhybridization at 42° C., and about 0.01 M to about 0.02 M salt forwashing at 55° C.

High stringency conditions also may include 1% BSA, 1 mM EDTA, 0.5 MNaHPO₄ (pH 7.2), 7% SDS for hybridization at 65° C., and (i) 0.2×SSC,0.1% SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS forwashing at a temperature in excess of 65° C. One embodiment of highstringency conditions includes hybridizing in 6×SSC at about 45° C.,followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C. Oneembodiment of very high stringency conditions includes hybridizing in0.5 M sodium phosphate, 7% SDS at 65° C., followed by one or more washesin 0.2×SSC, 1% SDS at 65° C.

Other stringency conditions are well known in the art and a skilledartisan will recognize that various factors can be manipulated tooptimize the specificity of the hybridization. Optimization of thestringency of the final washes can serve to ensure a high degree ofhybridization. For detailed examples, see Ausubel et al., supra at pages2.10.1 to 2.10.16 and Sambrook et al. (1989, supra) at sections 1.101 to1.104.

While stringent washes are typically carried out at temperatures fromabout 42° C. to 68° C., one skilled in the art will appreciate thatother temperatures may be suitable for stringent conditions. Maximumhybridization rate typically occurs at about 20° C. to 25° C. below theT_(m) for formation of a DNA-DNA hybrid. It is well known in the artthat the T_(m) is the melting temperature, or temperature at which twocomplementary polynucleotide sequences dissociate. Methods forestimating T_(m) are well known in the art (see Ausubel et al., supra atpage 2.10.8).

In general, the T_(m) of a perfectly matched duplex of DNA may bepredicted as an approximation by the formula: T_(m)=81.5+16.6 (log₁₀M)+0.41 (% G+C)−0.63 (% formamide)−(600/length) wherein: M is theconcentration of Na⁺, preferably in the range of 0.01 molar to 0.4molar; % G+C is the sum of guanosine and cytosine bases as a percentageof the total number of bases, within the range between 30% and 75% G+C;% formamide is the percent formamide concentration by volume; length isthe number of base pairs in the DNA duplex. The T_(m) of a duplex DNAdecreases by approximately 1° C. with every increase of 1% in the numberof randomly mismatched base pairs. Washing is generally carried out atT_(m)—15° C. for high stringency, or T_(m) —30° C. for moderatestringency.

In one example of a hybridization procedure, a membrane (e.g., anitrocellulose membrane or a nylon membrane) containing immobilized DNAis hybridized overnight at 42° C. in a hybridization buffer (50%deionized formamide, 5×SSC, 5×Denhardt's solution (0.1% ficoll, 0.1%polyvinylpyrollidone and 0.1% bovine serum albumin), 0.1% SDS and 200mg/mL denatured salmon sperm DNA) containing a labeled probe. Themembrane is then subjected to two sequential medium stringency washes(i.e., 2×SSC, 0.1% SDS for 15 min at 45° C., followed by 2×SSC, 0.1% SDSfor 15 min at 50° C.), followed by two sequential higher stringencywashes (i.e., 0.2×SSC, 0.1% SDS for 12 min at 55° C. followed by 0.2×SSCand 0.1% SDS solution for 12 min at 65-68° C.

As noted above, certain embodiments relate to AARS polynucleotides thatencode an AARS polypeptide. Among other uses, these embodiments may beutilized to recombinantly produce a desired AARS polypeptide or variantthereof, or to express the AARS polypeptide in a selected cell orsubject. It will be appreciated by those of ordinary skill in the artthat, as a result of the degeneracy of the genetic code, there are manynucleotide sequences that encode a polypeptide as described herein. Someof these polynucleotides may bear minimal homology to the nucleotidesequence of any native gene. Nonetheless, polynucleotides that vary dueto differences in codon usage are specifically contemplated by thepresent invention, for example polynucleotides that are optimized forhuman and/or primate codon selection.

Therefore, multiple polynucleotides can encode the AARS polypeptides ofthe invention. Moreover, the polynucleotide sequence can be manipulatedfor various reasons. Examples include but are not limited to theincorporation of preferred codons to enhance the expression of thepolynucleotide in various organisms (see generally Nakamura et al., Nuc.Acid. Res. (2000) 28 (1): 292). In addition, silent mutations can beincorporated in order to introduce, or eliminate restriction sites,decrease the density of CpG dinucleotide motifs (see for example, Kamedaet al., Biochem. Biophys. Res. Commun. (2006) 349(4): 1269-1277) orreduce the ability of single stranded sequences to form stem-loopstructures: (see, e.g., Zuker M., Nucl. Acid Res. (2003); 31(13):3406-3415). In addition, mammalian expression can be further optimizedby including a Kozak consensus sequence [i.e., (a/g)cc(a/g)ccATGg] atthe start codon. Kozak consensus sequences useful for this purpose areknown in the art (Mantyh et al. PNAS 92: 2662-2666 (1995); Mantyh et al.Prot. Exp. & Purif. 6,124 (1995)).

The polynucleotides of the present invention, regardless of the lengthof the coding sequence itself, may be combined with other DNA sequences,such as promoters, polyadenylation signals, additional restrictionenzyme sites, multiple cloning sites, other coding segments, and thelike, such that their overall length may vary considerably. It istherefore contemplated that a polynucleotide fragment of almost anylength may be employed; with the total length preferably being limitedby the ease of preparation and use in the intended recombinant DNAprotocol.

Polynucleotides and fusions thereof may be prepared, manipulated and/orexpressed using any of a variety of well established techniques knownand available in the art. For example, polynucleotide sequences whichencode polypeptides of the invention, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of an AARS polypeptide in appropriate host cells. Due to theinherent degeneracy of the genetic code, other DNA sequences that encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and express agiven polypeptide.

As will be understood by those of skill in the art, it may beadvantageous in some instances to produce polypeptide-encodingnucleotide sequences possessing non-naturally occurring codons. Forexample, codons preferred by a particular prokaryotic or eukaryotic hostcan be selected to increase the rate of protein expression or to producea recombinant RNA transcript having desirable properties, such as ahalf-life which is longer than that of a transcript generated from thenaturally occurring sequence. Such polynucleotides are commonly referredto as “codon-optimized.” Any of the polynucleotides described herein maybe utilized in a codon-optimized form. In certain embodiments, apolynucleotide can be codon optimized for use in specific bacteria suchas E. coli or yeast such as S. cerevisiae (see, e.g., Burgess-Brown etal., Protein Expr Purif. 59:94-102, 2008; Ermolaeva Md. (2001) Curr.Iss. Mol. Biol. 3 (4) 91-7; Welch et al., PLoS ONE 4(9): e7007doi:10.1371/journal.pone.0007002).

Moreover, the polynucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterpolypeptide encoding sequences for a variety of reasons, including butnot limited to, alterations which modify the cloning, processing,expression and/or activity of the gene product.

According to another aspect of the invention, polynucleotides encodingpolypeptides of the invention may be delivered to a subject in vivo,e.g., using gene therapy techniques. Gene therapy refers generally tothe transfer of heterologous nucleic acids to the certain cells, targetcells, of a mammal, particularly a human, with a disorder or conditionsfor which such therapy is sought. The nucleic acid is introduced intothe selected target cells in a manner such that the heterologous DNA isexpressed and a therapeutic product encoded thereby is produced.

Various viral vectors that can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, adeno-associatedvirus (AAV), or, preferably, an RNA virus such as a retrovirus.Preferably, the retroviral vector is a derivative of a murine or avianretrovirus, or is a lentiviral vector. The preferred retroviral vectoris a lentiviral vector. Examples of retroviral vectors in which a singleforeign gene can be inserted include, but are not limited to: Moloneymurine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV),murine mammary tumor virus (MuMTV), SIV, BIV, HIV and Rous Sarcoma Virus(RSV). A number of additional retroviral vectors can incorporatemultiple genes. All of these vectors can transfer or incorporate a genefor a selectable marker so that transduced cells can be identified andgenerated. By inserting a zinc finger derived-DNA binding polypeptidesequence of interest into the viral vector, along with another gene thatencodes the ligand for a receptor on a specific target cell, forexample, the vector may be made target specific. Retroviral vectors canbe made target specific by inserting, for example, a polynucleotideencoding a protein (dimer). Illustrative targeting may be accomplishedby using an antibody to target the retroviral vector. Those of skill inthe art will know of, or can readily ascertain without undueexperimentation, specific polynucleotide sequences which can be insertedinto the retroviral genome to allow target specific delivery of theretroviral vector containing the zinc finger-nucleotide binding proteinpolynucleotide.

Since recombinant retroviruses are defective, they require assistance inorder to produce infectious vector particles. This assistance can beprovided, for example, by using helper cell lines that contain plasmidsencoding all of the structural genes of the retrovirus under the controlof regulatory sequences within the LTR. These plasmids are missing anucleotide sequence which enables the packaging mechanism to recognizean RNA transcript for encapsulation. Helper cell lines which havedeletions of the packaging signal include but are not limited to PSI.2,PA317 and PA12, for example. These cell lines produce empty virions,since no genome is packaged. If a retroviral vector is introduced intosuch cells in which the packaging signal is intact, but the structuralgenes are replaced by other genes of interest, the vector can bepackaged and vector virion produced. The vector virions produced by thismethod can then be used to infect a tissue cell line, such as NIH 3T3cells, to produce large quantities of chimeric retroviral virions.

“Non-viral” delivery techniques for gene therapy can also be usedincluding, for example, DNA-ligand complexes, adenovirus-ligand-DNAcomplexes, direct injection of DNA, CaPO₄ precipitation, gene guntechniques, electroporation, liposomes, lipofection, and the like. Anyof these methods are widely available to one skilled in the art andwould be suitable for use in the present invention. Other suitablemethods are available to one skilled in the art, and it is to beunderstood that the present invention can be accomplished using any ofthe available methods of transfection. Lipofection can be accomplishedby encapsulating an isolated DNA molecule within a liposomal particleand contacting the liposomal particle with the cell membrane of thetarget cell. Liposomes are self-assembling, colloidal particles in whicha lipid bilayer, composed of amphiphilic molecules such as phosphatidylserine or phosphatidyl choline, encapsulates a portion of thesurrounding media such that the lipid bilayer surrounds a hydrophilicinterior. Unilammellar or multilammellar liposomes can be constructedsuch that the interior contains a desired chemical, drug, or, as in theinstant invention, an isolated DNA molecule.

In another aspect, polynucleotides encoding polypeptides of theinvention may be used to express and delivery an AARS polypeptide viacell therapy. Accordingly in another aspect, the current inventionincludes a cell therapy for treating a disease or disorder, comprisingadministering a host cell expressing, or capable of expressing, an AARSpolypeptide.

Cell therapy involves the administration of cells which have beenselected, multiplied and pharmacologically treated or altered (i.e.genetically modified) outside of the body (Bordignon, C. et al, CellTherapy: Achievements and Perspectives (1999), Haematologica, 84, pp.1110-1149). Such host cells include for example, primary cells,including macrophages, and stem cells which have been geneticallymodified to express an AARS polypeptide. The aim of cell therapy is toreplace, repair or enhance the biological function of damaged tissues ororgans.

The use of transplanted cells has been investigated for the treatment ofnumerous endocrine disorders such as anemia and dwarfism, hematologicaldisorders, kidney and liver failure, pituitary and CNS deficiencies anddiabetes mellitus (Uludag et al., Technology of Mammalian CellEncapsulation (2000), Advanced Drug Delivery Reviews, 42, pp. 29-64).Transplanted cells may function by releasing bioactive compounds such asan AARS polypeptide of the invention, to replace endogenous AARSpolypeptides which are absent or produced in insufficient quantities inan effected system.

Embodiments of the present invention also include oligonucleotides,whether for detection, amplification, antisense therapies, or otherpurpose. For these and related purposes, the term “oligonucleotide” or“oligo” or “oligomer” is intended to encompass a singular“oligonucleotide” as well as plural “oligonucleotides,” and refers toany polymer of two or more of nucleotides, nucleosides, nucleobases orrelated compounds used as a reagent in the amplification methods of thepresent invention, as well as subsequent detection methods. Theoligonucleotide may be DNA and/or RNA and/or analogs thereof.

The term oligonucleotide does not necessarily denote any particularfunction to the reagent, rather, it is used generically to cover allsuch reagents described herein. An oligonucleotide may serve variousdifferent functions, e.g., it may function as a primer if it is capableof hybridizing to a complementary strand and can further be extended inthe presence of a nucleic acid polymerase, it may provide a promoter ifit contains a sequence recognized by an RNA polymerase and allows fortranscription, and it may function to prevent hybridization or impedeprimer extension if appropriately situated and/or modified. Anoligonucleotide may also function as a probe, or an antisense agent. Anoligonucleotide can be virtually any length, limited only by itsspecific function, e.g., in an amplification reaction, in detecting anamplification product of the amplification reaction, or in an antisenseor RNA interference application. Any of the oligonucleotides describedherein can be used as a primer, a probe, an antisense oligomer, or anRNA interference agent.

The term “primer” as used herein refers to a single-strandedoligonucleotide capable of acting as a point of initiation fortemplate-directed DNA synthesis under suitable conditions defined, forexample, by buffer and temperature, in the presence of four differentnucleoside triphosphates and an agent for polymerization, such as a DNAor RNA polymerase or reverse transcriptase. The length of the primer, inany given case, depends on, for example, the intended use of the primer,and generally ranges from about 15 to 30 nucleotides, although shorterand longer primers may be used. Short primer molecules generally requirecooler temperatures to form sufficiently stable hybrid complexes withthe template. A primer need not reflect the exact sequence of thetemplate but must be sufficiently complementary to hybridize with suchtemplate. The primer site is the area of the template to which a primerhybridizes. The primer pair is a set of primers including a 5′ upstreamprimer that hybridizes with the 5′ end of the sequence to be amplifiedand a 3′ downstream primer that hybridizes with the complement of the 3′end of the sequence to be amplified.

The term “probe” as used herein includes a surface-immobilized orsoluble but capable of being immobilized molecule that can be recognizedby a particular target. See, e.g., U.S. Pat. No. 6,582,908 for anexample of arrays having all possible combinations of probes with 10,12, and more bases. Probes and primers as used herein typically compriseat least 10-15 contiguous nucleotides of a known sequence. In order toenhance specificity, longer probes and primers may also be employed,such as probes and primers that comprise at least 20, 25, 30, 40, 50,60, 70, 80, 90, 100, or at least 150 nucleotides of an AARS referencesequence or its complement. Probes and primers may be considerablylonger than these examples, and it is understood that any lengthsupported by the knowledge in the art and the specification, includingthe tables, figures, and Sequence Listing, may be used.

Methods for preparing and using probes and primers are described in thereferences, for example Sambrook, J. et al. (1989) Molecular Cloning: ALaboratory Manual, 2.sup.nd ed., vol. 1-3, Cold Spring Harbor Press,Plainview N.Y.; Ausubel, F. M. et al. (1987) Current Protocols inMolecular Biology, Greene Publ. Assoc. & Wiley-Intersciences, New YorkN.Y.; Innis, M. et al. (1990) PCR Protocols. A Guide to Methods andApplications, Academic Press, San Diego Calif. PCR primer pairs can bederived from a known sequence, for example, by using computer programsintended for that purpose such as Primer (Version 0.5, 1991, WhiteheadInstitute for Biomedical Research, Cambridge Mass.).

Oligonucleotides for use as primers or probes may be selected usingsoftware known in the art. For example, OLIGO 4.06 software is usefulfor the selection of PCR primer pairs of up to 100 nucleotides each, andfor the analysis of oligonucleotides and larger polynucleotides of up to5,000 nucleotides from an input polynucleotide sequence of up to 32kilobases. Similar primer selection programs have incorporatedadditional features for expanded capabilities. For example, the PrimOUprimer selection program (available to the public from the Genome Centerat University of Texas South West Medical Center, Dallas Tex.) iscapable of choosing specific primers from megabase sequences and is thususeful for designing primers on a genome-wide scope.

The Primer3 primer selection program (available to the public from theWhitehead Institute/MIT Center for Genome Research, Cambridge Mass.)allows the user to input a “mispriming library,” in which sequences toavoid as primer binding sites are user-specified. Primer3 is useful, inparticular, for the selection of oligonucleotides for microarrays. (Thesource code for the latter two primer selection programs may also beobtained from their respective sources and modified to meet the user'sspecific needs.) The PrimeGen program (available to the public from theUK Human Genome Mapping Project Resource Centre, Cambridge UK) designsprimers based on multiple sequence alignments, thereby allowingselection of primers that hybridize to either the most conserved orleast conserved regions of aligned nucleic acid sequences. Hence, thisprogram is useful for identification of both unique and conservedoligonucleotides and polynucleotide fragments. The oligonucleotides andpolynucleotide fragments identified by any of the above selectionmethods are useful in hybridization technologies, for example, as PCR orsequencing primers, microarray elements, or specific probes to identifyfully or partially complementary polynucleotides in a sample of nucleicacids. Methods of oligonucleotide selection are not limited to thosedescribed herein.

In certain embodiments, oligonucleotides can be prepared by stepwisesolid-phase synthesis, employing methods detailed in the referencescited above, and below with respect to the synthesis of oligonucleotideshaving a mixture or uncharged and cationic backbone linkages. In somecases, it may be desirable to add additional chemical moieties to theoligonucleotide, e.g., to enhance pharmacokinetics or to facilitatecapture or detection of the compound. Such a moiety may be covalentlyattached, typically to a terminus of the oligomer, according to standardsynthetic methods. For example, addition of a polyethyleneglycol moietyor other hydrophilic polymer, e.g., one having 10-100 monomericsubunits, may be useful in enhancing solubility. One or more chargedgroups, e.g., anionic charged groups such as an organic acid, mayenhance cell uptake.

A variety of detectable molecules may be used to render anoligonucleotide, or protein detectable, such as a radioisotopes,fluorochromes, dyes, enzymes, nanoparticles, chemiluminescent markers,biotin, or other monomer known in the art that can be detected directly(e.g., by light emission) or indirectly (e.g., by binding of afluorescently-labeled antibody).

Radioisotopes provide examples of detectable molecules that can beutilized in certain aspects of the present invention. Severalradioisotopes can be used as detectable molecules for labelingnucleotides or proteins, including, for example, ³²P, ³³P, ³⁵S, ³H, and¹²⁵I. These radioisotopes have different half-lives, types of decay, andlevels of energy which can be tailored to match the needs of aparticular protocol. For example, ³H is a low energy emitter whichresults in low background levels, however this low energy also resultsin long time periods for autoradiography. Radioactively labeledribonucleotides, deoxyribonucleotides and amino acids are commerciallyavailable. Nucleotides are available that are radioactively labeled atthe first, or α, phosphate group, or the third, or γ, phosphate group.For example, both [α-³²P] dATP and [γ-³²P] dATP are commerciallyavailable. In addition, different specific activities for radioactivelylabeled nucleotides are also available commercially and can be tailoredfor different protocols.

Other examples of detectable molecules that can be utilized to detect anoligonucleotide include fluorophores. Several fluorophores can be usedfor labeling nucleotides including, for example, fluorescein,tetramethylrhodamine, Texas Red, and a number of others (e.g., Haugland,Handbook of Fluorescent Probes—9th Ed., 2002, Molec. Probes, Inc.,Eugene Oreg.; Haugland, The Handbook: A Guide to Fluorescent Probes andLabeling Technologies—10th Ed., 2005, Invitrogen, Carlsbad, Calif.).

As one example, oligonucleotides may be fluorescently labeled duringchemical synthesis, since incorporation of amines or thiols duringnucleotide synthesis permit addition of fluorophores. Fluorescentlylabeled nucleotides are commercially available. For example, uridine anddeoxyuridine triphosphates are available that are conjugated to tendifferent fluorophores that cover the spectrum. Fluorescent dyes thatcan be bound directly to nucleotides can also be utilized as detectablemolecules. For example, FAM, JOE, TAMRA, and ROX are amine reactivefluorescent dyes that have been attached to nucleotides and are used inautomated DNA sequencing. These fluorescently labeled nucleotides, forexample, ROX-ddATP, ROX-ddCTP, ROX-ddGTP and ROX-ddUTP, are commerciallyavailable.

Non-radioactive and non-fluorescent detectable molecules are alsoavailable. As noted above, biotin can be attached directly tonucleotides and detected by specific and high affinity binding to avidinor streptavidin which has been chemically coupled to an enzymecatalyzing a colorimetric reaction (such as phosphatase, luciferase, orperoxidase). Digoxigenin labeled nucleotides can also similarly be usedfor non-isotopic detection of nucleic acids. Biotinylated anddigoxigenin-labeled nucleotides are commercially available.

Very small particles, termed nanoparticles, also can be used to labeloligonucleotide probes. These particles range from 1-1000 nm in size andinclude diverse chemical structures such as gold and silver particlesand quantum dots. When irradiated with angled incident white light,silver or gold nanoparticles ranging from 40-120 nm will scattermonochromatic light with high intensity. The wavelength of the scatteredlight is dependent on the size of the particle. Four to five differentparticles in close proximity will each scatter monochromatic light,which when superimposed will give a specific, unique color. Theparticles are being manufactured by companies such as Genicon Sciences(Carlsbad, Calif.). Derivatized silver or gold particles can be attachedto a broad array of molecules including, proteins, antibodies, smallmolecules, receptor ligands, and nucleic acids. For example, the surfaceof the particle can be chemically derivatized to allow attachment to anucleotide.

Other types of nanoparticles that can be used for detection of adetectable molecule include quantum dots. Quantum dots are fluorescingcrystals 1-5 nm in diameter that are excitable by light over a largerange of wavelengths. Upon excitation by light having an appropriatewavelength, these crystals emit light, such as monochromatic light, witha wavelength dependent on their chemical composition and size. Quantumdots such as CdSe, ZnSe, InP, or InAs possess unique optical properties;these and similar quantum dots are available from a number of commercialsources (e.g., NN-Labs, Fayetteville, Ark.; Ocean Nanotech,Fayetteville, Ark.; Nanoco Technologies, Manchester, UK; Sigma-Aldrich,St. Louis, Mo.).

Many dozens of classes of particles can be created according to thenumber of size classes of the quantum dot crystals. The size classes ofthe crystals are created either 1) by tight control of crystal formationparameters to create each desired size class of particle, or 2) bycreation of batches of crystals under loosely controlled crystalformation parameters, followed by sorting according to desired sizeand/or emission wavelengths. Two examples of references in which quantumdots are embedded within intrinsic silicon epitaxial layers ofsemiconductor light emitting/detecting devices are U.S. Pat. Nos.5,293,050 and 5,354,707 to Chapple Sokol, et al.

In certain embodiments, oligonucleotide primers or probes may be labeledwith one or more light-emitting or otherwise detectable dyes. The lightemitted by the dyes can be visible light or invisible light, such asultraviolet or infrared light. In exemplary embodiments, the dye may bea fluorescence resonance energy transfer (FRET) dye; a xanthene dye,such as fluorescein and rhodamine; a dye that has an amino group in thealpha or beta position (such as a naphthylamine dye,1-dimethylaminonaphthyl-5-sulfonate, 1-anilino-8-naphthalende sulfonateand 2-p-touidinyl-6-naphthalene sulfonate); a dye that has3-phenyl-7-isocyanatocoumarin; an acridine, such as9-isothiocyanatoacridine and acridine orange; a pyrene, a bensoxadiazoleand a stilbene; a dye that has3-(ε-carboxypentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CYA);6-carboxy fluorescein (FAM); 5&6-carboxyrhodamine-110 (R110);6-carboxyrhodamine-6G (R6G); N,N,N′,N′-tetramethyl-6-carboxyrhodamine(TAMRA); 6-carboxy-X-rhodamine (ROX);6-carboxy-4′,5′-dichloro-2′,7′-dimethoxyfluorescein (JOE); ALEXA FLUOR™;Cy2; Texas Red and Rhodamine Red;6-carboxy-2′,4,7,7′-tetrachlorofluorescein (TET);6-carboxy-2′,4,4′,5′,7,7′-hexachlorofluorescein (HEX); 5-carboxy-2′,4′,5‘,7’-tetrachlorofluorescein (ZOE); NAN; NED; Cy3; Cy3.5; Cy5; Cy5.5;Cy7; and Cy7.5; IR800CW, ICG, Alexa Fluor 350; Alexa Fluor 488; AlexaFluor 532; Alexa Fluor 546; Alexa Fluor 568; Alexa Fluor 594; AlexaFluor 647; Alexa Fluor 680, or Alexa Fluor 750.

The AARS polynucleotides and oligonucleotides of the present inventioncan be used in any of the therapeutic, diagnostic, research, or drugdiscovery compositions and methods described herein.

V. ANTIBODIES

According to another aspect, the present invention further providesantibodies that exhibit binding specificity for an AARS polypeptide, orits native cellular binding partner (i.e. cellular receptor, lipid,carbohydrate, protein, or nucleic acid binding partner), or complexthereof, and methods of using the same. The term antibody includes thevarious variations of the same, such as FABs, human antibodies, modifiedhuman antibodies, single chains, nonhuman antibodies, and otherderivatives of the immunoglobulin fold that underlie immune systemligands for antigens, as described herein and known in the art.Antibodies can be used in any of the therapeutic, diagnostic, drugdiscovery, or protein expression/purification methods and compositionsprovided herein.

Certain antibodies of the present invention differ from certainpreviously made antibodies because they can distinguish between the AARSprotein fragments of Table(s) 1-3 and their corresponding full-lengthAARS, typically by binding with greater affinity to the AARS proteinfragments than to the corresponding full-length AARS. Generally, suchantibodies may bind to unique sequences or structures generated orrevealed by splice variations, proteolysis, or other cellular processingthat generates an AARS protein fragment of the invention (e.g., posttranslational processing, including but not limited to phosphorylationand other modifications that change protein structure). In some aspectsthe antibodies may bind to sequences around a unique splice junction(for example to one or more regions of at least 5 contiguous amino acidsselected from the splice junction sequences listed in Table 2B, oralternatively to any amino acid sequence C-terminal of this splice site,for example as listed in Table 2B. For example, such antibodies may havebinding specificity to one or more non-solvent exposed faces that areexposed in the AARS protein fragment but not in the full-length AARS, orsequences that are not found or are otherwise inaccessible in thefull-length AARS. Antibodies may also bind to unique three-dimensionalstructures that result from differences in folding between the AARSprotein fragment and the full-length AARS. Such differences in foldingmay be localized (e.g., to a specific domain or region) or globalized.As one example, folding of AARS protein fragments may generate uniquecontinuous or discontinuous epitopes that are not found in thecorresponding or parent AARS. Examples also include antibodies thatspecifically bind to N- or C-termini generated by splice variations,proteolysis, or other cellular processing; such termini may be uniquecompared to the full-length AARS or may not be exposed for antibodybinding in the full-length versions due to their termini beingcompletely or partially buried in the overall structure of the largerAARS parent molecule.

In some embodiments, antibodies provided herein do not form aggregates,have a desired solubility, and/or have an immunogenicity profile that issuitable for use in humans, as described herein and known in the art.Also included are antibodies that are suitable for production work, suchas to purify the AARS protein fragments described herein. Preferably,active antibodies can be concentrated to at least about 10 mg/ml andoptional formulated for biotherapeutic uses.

In certain embodiments, antibodies are effective for modulating one ormore of the non-canonical activities mediated by an AARS polypeptide ofthe invention. In certain embodiments, for example, the antibody is onethat binds to an AARS polypeptide and/or its binding partner, inhibitstheir ability to interact with each other, and/or antagonizes thenon-canonical activity of the AARS polypeptide. In certain embodiments,for example, the antibody binds to the cellular binding partner of anAARS polypeptide, and mimics the AARS polypeptide activity, such as byincreasing or agonizing the non-canonical activity mediated by the AARSpolypeptide. Accordingly, antibodies may be used to diagnose, treat, orprevent diseases, disorders or other conditions that are mediated by anAARS polypeptide of the invention, such as by antagonizing or agonizingits activity partially or fully.

An antibody, or antigen-binding fragment thereof, is said to“specifically bind,” “immunologically bind,” and/or is “immunologicallyreactive” to a polypeptide of the invention if it reacts at a detectablelevel (within, for example, an ELISA assay) with the polypeptide, anddoes not react detectably in a statistically significant manner withunrelated polypeptides under similar conditions. In certain instances, abinding agent does not significantly interact with a full-length versionof the AARS polypeptide.

Immunological binding, as used in this context, generally refers to thenon-covalent interactions of the type which occur between animmunoglobulin molecule and an antigen for which the immunoglobulin isspecific. The strength, or affinity of binding such as immunologicalbinding interactions can be expressed in terms of the dissociationconstant (K_(d)) of the interaction, wherein a smaller K_(d) representsa greater affinity. Immunological binding properties of selectedpolypeptides can be quantified using methods well known in the art. See,e.g., Davies et al. (1990) Annual Rev. Biochem. 59:439-473. In certainillustrative embodiments, an antibody has an affinity for an AARSprotein fragment of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5,0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50nM. In certain embodiments, the affinity of the antibody for an AARSprotein fragment is stronger than its affinity for a correspondingfull-length AARS polypeptide, typically by about 1.5×, 2×, 2.5×, 3×,3.5×, 4×, 4.5×, 5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×, 30×, 40×, 50×,60×, 70×, 80×, 90×, 100×, 200×, 300×, 400×, 500×, 600×, 700×, 800×,900×, 1000× or more (including all integers in between). In certainembodiments, an antibody as an affinity for a corresponding full-lengthAARS protein of at least about 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 μM. Incertain embodiments, an antibody binds weakly or substantiallyundetectably to a full-length AARS protein.

An “antigen-binding site,” or “binding portion” of an antibody, refersto the part of the immunoglobulin molecule that participates in antigenbinding. The antigen binding site is formed by amino acid residues ofthe N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”)chains. Three highly divergent stretches within the V regions of theheavy and light chains are referred to as “hypervariable regions” whichare interposed between more conserved flanking stretches known as“framework regions,” or “FRs”. Thus the term “FR” refers to amino acidsequences which are naturally found between and adjacent tohypervariable regions in immunoglobulins. In an antibody molecule, thethree hypervariable regions of a light chain and the three hypervariableregions of a heavy chain are disposed relative to each other in threedimensional space to form an antigen-binding surface. Theantigen-binding surface is complementary to the three-dimensionalsurface of a bound antigen, and the three hypervariable regions of eachof the heavy and light chains are referred to as“complementarity-determining regions,” or “CDRs.”

Antibodies may be prepared by any of a variety of techniques known tothose of ordinary skill in the art. See, e.g., Harlow and Lane,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988.Monoclonal antibodies specific for a polypeptide of interest may beprepared, for example, using the technique of Kohler and Milstein, Eur.J. Immunol. 6:511-519, 1976, and improvements thereto. Also included aremethods that utilize transgenic animals such as mice to express humanantibodies. See, e.g., Neuberger et al., Nature Biotechnology 14:826,1996; Lonberg et al., Handbook of Experimental Pharmacology 113:49-101,1994; and Lonberg et al., Internal Review of Immunology 13:65-93, 1995.Particular examples include the VELOCIMMUNE® platform by REGERNEREX®(see, e.g., U.S. Pat. No. 6,596,541). Antibodies can also be generatedor identified by the use of phage display or yeast display libraries(see, e.g., U.S. Pat. No. 7,244,592; Chao et al., Nature Protocols.1:755-768, 2006). Non-limiting examples of available libraries includecloned or synthetic libraries, such as the Human Combinatorial AntibodyLibrary (HuCAL), in which the structural diversity of the human antibodyrepertoire is represented by seven heavy chain and seven light chainvariable region genes. The combination of these genes gives rise to 49frameworks in the master library. By superimposing highly variablegenetic cassettes (CDRs=complementarity determining regions) on theseframeworks, the vast human antibody repertoire can be reproduced. Alsoincluded are human libraries designed with human-donor-sourced fragmentsencoding a light-chain variable region, a heavy-chain CDR-3, syntheticDNA encoding diversity in heavy-chain CDR-1, and synthetic DNA encodingdiversity in heavy-chain CDR-2. Other libraries suitable for use will beapparent to persons skilled in the art. The polypeptides of thisinvention may be used in the purification process in, for example, anaffinity chromatography step.

An “Fv” fragment can be produced by preferential proteolytic cleavage ofan IgM, and on rare occasions IgG or IgA immunoglobulin molecule. Fvfragments are, however, more commonly derived using recombinanttechniques known in the art. The Fv fragment includes a non-covalentV_(H)::V_(L) heterodimer including an antigen-binding site which retainsmuch of the antigen recognition and binding capabilities of the nativeantibody molecule. See, e.g., Inbar et al. (1972) Proc. Nat. Acad. Sci.USA 69:2659-2662; Hochman et al. (1976) Biochem 15:2706-2710; andEhrlich et al. (1980) Biochem 19:4091-4096.

A single chain Fv (“sFv”) polypeptide is a covalently linkedV_(H)::V_(L) heterodimer which is expressed from a gene fusion includingV_(H)- and V_(L)-encoding genes linked by a peptide-encoding linker.Huston et al. (1988) PNAS USA. 85(16):5879-5883. A number of methodshave been described to discern chemical structures for converting thenaturally aggregated—but chemically separated—light and heavypolypeptide chains from an antibody V region into an sFv molecule whichwill fold into a three dimensional structure substantially similar tothe structure of an antigen-binding site. See, e.g., U.S. Pat. Nos.5,091,513 and 5,132,405, to Huston et al.; and U.S. Pat. No. 4,946,778,to Ladner et al.

Each of the above-described molecules includes a heavy chain and a lightchain CDR set, respectively interposed between a heavy chain and a lightchain FR set which provide support to the CDRS and define the spatialrelationship of the CDRs relative to each other. As used herein, theterm “CDR set” refers to the three hypervariable regions of a heavy orlight chain V region. Proceeding from the N-terminus of a heavy or lightchain, these regions are denoted as “CDR1,” “CDR2,” and “CDR3”respectively. An antigen-binding site, therefore, includes six CDRs,comprising the CDR set from each of a heavy and a light chain V region.A polypeptide comprising a single CDR, (e.g., a CDR1, CDR2 or CDR3) isreferred to herein as a “molecular recognition unit.” Crystallographicanalysis of a number of antigen-antibody complexes has demonstrated thatthe amino acid residues of CDRs form extensive contact with boundantigen, wherein the most extensive antigen contact is with the heavychain CDR3. Thus, the molecular recognition units are primarilyresponsible for the specificity of an antigen-binding site.

As used herein, the term “FR set” refers to the four flanking amino acidsequences which frame the CDRs of a CDR set of a heavy or light chain Vregion. Some FR residues may contact bound antigen; however, FRs areprimarily responsible for folding the V region into the antigen-bindingsite, particularly the FR residues directly adjacent to the CDRS. WithinFRs, certain amino residues and certain structural features are veryhighly conserved. In this regard, all V region sequences contain aninternal disulfide loop of around 90 amino acid residues. When the Vregions fold into a binding-site, the CDRs are displayed as projectingloop motifs which form an antigen-binding surface. It is generallyrecognized that there are conserved structural regions of FRs whichinfluence the folded shape of the CDR loops into certain “canonical”structures—regardless of the precise CDR amino acid sequence. Further,certain FR residues are known to participate in non-covalent interdomaincontacts which stabilize the interaction of the antibody heavy and lightchains.

Certain embodiments include single domain antibody (sdAbs or“nanobodies”), which refer to an antibody fragment consisting of asingle monomeric variable antibody domain (see, e.g., U.S. Pat. Nos.5,840,526; 5,874,541; 6,005,079, 6,765,087, 5,800,988; 5,874,541; and6,015,695). Such sdABs typically have a molecular weight of about 12-15kDa. In certain aspects, sdABs and other antibody molecules can bederived or isolated from the unique heavy-chain antibodies of immunizedcamels and llamas, often referred to as camelids. See, e.g., Conrath etal., JBC. 276:7346-7350, 2001.

A number of “humanized” antibody molecules comprising an antigen-bindingsite derived from a non-human immunoglobulin have been described,including chimeric antibodies having rodent V regions and theirassociated CDRs fused to human constant domains (Winter et al. (1991)Nature 349:293-299; Lobuglio et al. (1989) Proc. Nat. Acad. Sci. USA86:4220-4224; Shaw et al. (1987) J Immunol. 138:4534-4538; and Brown etal. (1987) Cancer Res. 47:3577-3583), rodent CDRs grafted into a humansupporting FR prior to fusion with an appropriate human antibodyconstant domain (Riechmann et al. (1988) Nature 332:323-327; Verhoeyenet al. (1988) Science 239:1534-1536; and Jones et al. (1986) Nature321:522-525), and rodent CDRs supported by recombinantly veneered rodentFRs (European Patent Publication No. 519,596, published Dec. 23, 1992).These “humanized” molecules are designed to minimize unwantedimmunological response toward rodent antihuman antibody molecules whichlimits the duration and effectiveness of therapeutic applications ofthose moieties in human recipients. See, e.g., U.S. Pat. Nos. 5,530,101;5,585,089; 5,693,762; 6,180,370; and 7,022,500.

The antibodies of the present invention can be used in any of thetherapeutic, diagnostic, drug discovery, protein purification, andanalytical methods and compositions described herein.

VI. ANTIBODY ALTERNATIVES AND OTHER BINDING AGENTS

According to another aspect, the present invention further providesantibody alternatives or other binding agents, such as solublereceptors, adnectins, peptides, peptide mimetics, small molecules,aptamers, etc., that exhibit binding specificity for an AARS polypeptideor its cellular binding partner as disclosed herein, or to a portion,variant or derivative thereof, and compositions and methods of usingsame. Binding agents can be used in any of the therapeutic, diagnostic,drug discovery, or protein expression/purification, and analyticalmethods and compositions described herein. Biologic-based binding agentssuch as adnectins, soluble receptors, avimers, and trinectins areparticularly useful.

In certain embodiments, such binding agents are effective for modulatingone or more of the non-canonical activities mediated by an AARSpolypeptide of the invention. In some embodiments, for example, thebinding agent is one that binds to an AARS polypeptide and/or itsbinding partner, inhibits their ability to interact with each other,and/or antagonizes the non-canonical activity of the AARS polypeptide.In certain embodiments, for example, the binding agent binds to thecellular binding partner of an AARS polypeptide, and mimics the AARSpolypeptide activity, such as by increasing or agonizing thenon-canonical activity mediated by the AARS polypeptide. Accordingly,such binding agents may be used to diagnose, treat, or prevent diseases,disorders or other conditions that are mediated by an AARS polypeptideof the invention, such as by antagonizing or agonizing its activitypartially or fully.

A binding agent is said to “specifically bind” to an AARS polypeptide ofthe invention, or its cellular binding partner, if it reacts at adetectable level (within, for example, an ELISA assay) with thepolypeptide or its cellular binding partner, and does not reactdetectably in a statistically significant manner with unrelatedpolypeptides under similar conditions. In certain instances, a bindingagent does not significantly interact with a full-length version of theAARS polypeptide. In certain illustrative embodiments, a binding agenthas an affinity for an AARS protein fragment or its cellular bindingpartner of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50 nM. In certainembodiments, the affinity of the binding agent for an AARS proteinfragment is stronger than its affinity for a corresponding full-lengthAARS polypeptide, typically by about 1.5×, 2×, 2.5×, 3×, 3.5×, 4×, 4.5×,5×, 6×, 7×, 8×, 9×, 10×, 15×, 20×, 25×, 30×, 40×, 50×, 60×, 70×, 80×,90×, 100×, 200×, 300×, 400×, 500×, 600×, 700×, 800×, 900×, 1000× or more(including all integers in between). In certain embodiments, a bindingagent has an affinity for a corresponding full-length AARS protein of atleast about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, or 20 μM.

As noted above, “peptides” are included as binding agents. The termpeptide typically refers to a polymer of amino acid residues and tovariants and synthetic analogues of the same. In certain embodiments,the term “peptide” refers to relatively short polypeptides, includingpeptides that consist of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 amino acids,including all integers and ranges (e.g., 5-10, 8-12, 10-15) in between,and interact with an AARS polypeptide, its cellular binding partner, orboth. Peptides can be composed of naturally-occurring amino acids and/ornon-naturally occurring amino acids, as described herein.

In addition to peptides consisting only of naturally-occurring aminoacids, peptidomimetics or peptide analogs are also provided. Peptideanalogs are commonly used in the pharmaceutical industry as non-peptidedrugs with properties analogous to those of the template peptide. Thesetypes of non-peptide compound are termed “peptide mimetics” or“peptidomimetics” (Luthman, et al., A Textbook of Drug Design andDevelopment, 14:386-406, 2nd Ed., Harwood Academic Publishers (1996);Joachim Grante, Angew. Chem. Int. Ed. Engl., 33:1699-1720 (1994);Fauchere, J., Adv. Drug Res., 15:29 (1986); Veber and Freidinger TINS,p. 392 (1985); and Evans, et al., J. Med. Chem. 30:229 (1987)). Apeptidomimetic is a molecule that mimics the biological activity of apeptide but is no longer peptidic in chemical nature. Peptidomimeticcompounds are known in the art and are described, for example, in U.S.Pat. No. 6,245,886.

The present invention also includes peptoids. Peptoid derivatives ofpeptides represent another form of modified peptides that retain theimportant structural determinants for biological activity, yet eliminatethe peptide bonds, thereby conferring resistance to proteolysis (Simon,et al., PNAS USA. 89:9367-9371, 1992). Peptoids are oligomers ofN-substituted glycines. A number of N-alkyl groups have been described,each corresponding to the side chain of a natural amino acid. Thepeptidomimetics of the present invention include compounds in which atleast one amino acid, a few amino acids or all amino acid residues arereplaced by the corresponding N-substituted glycines. Peptoid librariesare described, for example, in U.S. Pat. No. 5,811,387.

A binding agent may also include one or more small molecules. A “smallmolecule” refers to an organic compound that is of synthetic orbiological origin (biomolecule), but is typically not a polymer. Organiccompounds refer to a large class of chemical compounds whose moleculescontain carbon, typically excluding those that contain only carbonates,simple oxides of carbon, or cyanides. A “biomolecule” refers generallyto an organic molecule that is produced by a living organism, includinglarge polymeric molecules (biopolymers) such as peptides,polysaccharides, and nucleic acids as well, and small molecules such asprimary secondary metabolites, lipids, phospholipids, glycolipids,sterols, glycerolipids, vitamins, and hormones. A “polymer” refersgenerally to a large molecule or macromolecule composed of repeatingstructural units, which are typically connected by covalent chemicalbond.

In certain embodiments, a small molecule has a molecular weight of lessthan 1000-2000 Daltons, typically between about 300 and 700 Daltons, andincluding about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,500, 650, 600, 750, 700, 850, 800, 950, 1000 or 2000 Daltons. Smallmolecule libraries are described elsewhere herein.

Aptamers are also included as binding agents (see, e.g., Ellington etal., Nature. 346, 818-22, 1990; and Tuerk et al., Science. 249, 505-10,1990). Examples of aptamers included nucleic acid aptamers (e.g., DNAaptamers, RNA aptamers) and peptide aptamers. Nucleic acid aptamersrefer generally to nucleic acid species that have been engineeredthrough repeated rounds of in vitro selection or equivalent method, suchas SELEX (systematic evolution of ligands by exponential enrichment), tobind to various molecular targets such as small molecules, proteins,nucleic acids, and even cells, tissues and organisms. See, e.g., U.S.Pat. Nos. 6,376,190; and 6,387,620. Hence, included are nucleic acidaptamers that bind to the AARS polypeptides described herein and/ortheir cellular binding partners.

Peptide aptamers typically include a variable peptide loop attached atboth ends to a protein scaffold, a double structural constraint thattypically increases the binding affinity of the peptide aptamer tolevels comparable to that of an antibody's (e.g., in the nanomolarrange). In certain embodiments, the variable loop length may be composedof about 10-20 amino acids (including all integers in between), and thescaffold may include any protein that has good solubility and compacityproperties. Certain exemplary embodiments may utilize the bacterialprotein Thioredoxin-A as a scaffold protein, the variable loop beinginserted within the reducing active site (-Cys-Gly-Pro-Cys- loop in thewild protein), with the two cysteines lateral chains being able to forma disulfide bridge. Methods for identifying peptide aptamers aredescribed, for example, in U.S. Application No. 2003/0108532. Hence,included are peptide aptamers that bind to the AARS polypeptidesdescribed herein and/or their cellular binding partners. Peptide aptamerselection can be performed using different systems known in the art,including the yeast two-hybrid system.

Also included are ADNECTINS™, AVIMERS™, anaphones and anticalins thatspecifically bind to an AARS protein fragment of the invention.ADNECTINS™ refer to a class of targeted biologics derived from humanfibronectin, an abundant extracellular protein that naturally binds toother proteins. See, e.g., U.S. Application Nos. 2007/0082365;2008/0139791; and 2008/0220049. ADNECTINS™ typically consists of anatural fibronectin backbone, as well as the multiple targeting domainsof a specific portion of human fibronectin. The targeting domains can beengineered to enable an ADNECTIN™ to specifically recognize atherapeutic target of interest, such as an AARS protein fragment of theinvention.

AVIMERS™ refer to multimeric binding proteins or peptides engineeredusing in vitro exon shuffling and phage display. Multiple bindingdomains are linked, resulting in greater affinity and specificitycompared to single epitope immunoglobulin domains. See, e.g., Silvermanet al., Nature Biotechnology. 23:1556-1561, 2005; U.S. Pat. No.7,166,697; and U.S. Application Nos. 2004/0175756, 2005/0048512,2005/0053973, 2005/0089932 and 2005/0221384.

Also included are designed ankyrin repeat proteins (DARPins), whichinclude a class of non-immunoglobulin proteins that can offer advantagesover antibodies for target binding in drug discovery and drugdevelopment. Among other uses, DARPins are ideally suited for in vivoimaging or delivery of toxins or other therapeutic payloads because oftheir favorable molecular properties, including small size and highstability. The low-cost production in bacteria and the rapid generationof many target-specific DARPins make the DARPin approach useful for drugdiscovery. Additionally, DARPins can be easily generated inmultispecific formats, offering the potential to target an effectorDARPin to a specific organ or to target multiple receptors with onemolecule composed of several DARPins. See, e.g., Stumpp et al., CurrOpin Drug Discov Devel. 10:153-159, 2007; U.S. Application No.2009/0082274; and PCT/EP2001/10454.

Certain embodiments include “monobodies,” which typically utilize the10th fibronectin type III domain of human fibronectin (FNfn10) as ascaffold to display multiple surface loops for target binding. FNfn10 isa small (94 residues) protein with a β-sandwich structure similar to theimmunoglobulin fold. It is highly stable without disulfide bonds ormetal ions, and it can be expressed in the correctly folded form at ahigh level in bacteria. The FNfn10 scaffold is compatible with virtuallyany display technologies. See, e.g., Batori et al., Protein Eng.15:1015-20, 2002; and Wojcik et al., Nat Struct Mol Biol., 2010; andU.S. Pat. No. 6,673,901.

Anticalins refer to a class of antibody mimetics, which are typicallysynthesized from human lipocalins, a family of binding proteins with ahypervariable loop region supported by a structurally rigid framework.See, e.g., U.S. Application No. 2006/0058510. Anticalins typically havea size of about 20 kDa. Anticalins can be characterized by a barrelstructure formed by eight antiparallel β-strands (a stable β-barrelscaffold) that are pairwise connected by four peptide loops and anattached α-helix. In certain aspects, conformational deviations toachieve specific binding are made in the hypervariable loop region(s).See, e.g., Skerra, FEBS J. 275:2677-83, 2008, herein incorporated byreference.

VII. BIOASSAYS AND ANALYTICAL ASSAYS FOR DRUG RELEASE ASSAYS AND PRODUCTSPECIFICATIONS, DIAGNOSTICS, AND REAGENTS

Also included are bioassays that relate to the AARS protein fragmentsand related agents as therapeutic and diagnostic reagents. Examplesinclude bioassays and analytical assays that measure purity, biologicalactivity, affinity, solubility, pH, endotoxin levels, among others, manyof which are described herein. Also included are assays that establishdose response curves and/or provide one or more bases for comparisonbetween different batches of agents. Batch comparisons can be based onany one or more of chemical characterization, biologicalcharacterization, and clinical characterization. For protein agents,also included are methods of evaluating the potency, stability,pharmacokinetics, and immunogenicity of a selected agent. Among otheruses, these and other methods can be used for lot releasing testing ofbiologic or chemical agents, including the AARS protein fragments,antibodies, binding agents, polynucleotides such as antisense agents andvectors, and others described herein.

Certain embodiments include the use of bioaffinity assays. Such assayscan be used to assess the binding affinity, for example, between an AARSprotein fragment and a cellular binding partner, or between an AARSprotein fragment and an antibody. Binding affinity can also be measuredbetween an AARS protein fragment and an alternate binding agent such asa candidate or lead test compound (e.g., small molecule modulator of anAARS), or between an AARS cellular binding partner and a candidate orlead test compound. Certain exemplary binding affinity assays mayutilize ELISA assays, as described herein and known in the art. Certainassays utilize high-performance receptor binding chromatography (see,e.g., Roswall et al., Biologicals. 24:25-39, 1996). Other exemplarybinding affinity assays may utilize surface plasmon resonance(SPR)-based technologies. Examples include BIACore technologies, certainof which integrate SPR technology with a microfluidics system to monitormolecular interactions in real time at concentrations ranging from pM tomM. Also included are KINEXA™ assays, which provide accuratemeasurements of binding specificity, binding affinity, and bindingkinetics/rate constants.

Certain embodiments relate to immunoassays for evaluating or optimizingthe immunogenicity of protein agents. Examples include ex vivo humancellular assays and in vitro immuno-enzymatic assays to provide usefulinformation on the immunogenic potential of a therapeutic protein. Exvivo cell-response assays can be used, for example, to reproduce thecellular co-operation between antigen-presenting cells (APCs) andT-cells, and thereby measure T-cells activation after contact with aprotein of interest. Certain in vitro enzymatic assays may utilize acollection of recombinant HLA-DR molecules that cover a significantportion of a relevant human population, and may include automatedimmuno-enzymatic assays for testing the binding of peptides (stemmingfrom the fragmentation of the therapeutic protein) with the HLA-DRmolecules. Also included are methods of reducing the immunogenicity of aselected protein, such as by using these and related methods to identifyand then remove or alter one or more T-cell epitopes from a proteinagent.

Also included are biological release assays (e.g., cell-based assays)for measuring parameters such as specific biological activities,including non-canonical biological activities, and cytotoxicity. Certainspecific biological assays include, for example, cell-based assays thatutilize a cellular binding partner (e.g., cell-surface receptor) of aselected AARS protein fragment, which is functionally coupled to areadout, such as a fluorescent or luminescent indicator of anon-canonical biological activity, as described herein. For instance,specific embodiments include a cell that comprises a cell-surfacereceptor or an extracellular portion thereof that binds to an AARSprotein fragment, wherein the cell comprises a detector or readout. Alsoincluded are in vivo biological assays to characterize thepharmacokinetics of an agent, such as an AARS polypeptide or antibody,typically utilizing engineered mice or other mammal (see, e.g., Lee etal., The Journal of Pharmacology. 281:1431-1439, 1997). Examples ofcytotoxicity-based biological assays include release assays (e.g.,chromium or europium release assays to measure apoptosis; see, e.g., vonZons et al., Clin Diagn Lab Immunol. 4:202-207, 1997), among others,which can assess the cytotoxicity of AARS protein fragments, whether forestablishing dose response curves, batch testing, or other propertiesrelated to approval by various regulatory agencies, such as the Food andDrug Administration (FDA).

Such assays can be used, for example, to develop a dose response curvefor a selected AARS protein fragment or other agent, and/or to comparethe dose response curve of different batches of proteins or otheragents. A dose-response curve is an X-Y graph that relates the magnitudeof a stressor to the response of a receptor; the response may be aphysiological or biochemical response, such as a non-canonicalbiological activity in a cell in vitro or in a cell or tissue in vivo, atherapeutically effective amount as measured in vivo (e.g., as measuredby EC₅₀), or death, whether measured in vitro or in vivo (e.g., celldeath, organismal death). Death is usually indicated as an LD₅₀, astatistically-derived dose that is lethal to 50% of a modeledpopulation, though it can be indicated by LC₀₁ (lethal dose for 1% ofthe animal test population), LC₁₀₀ (lethal dose for 100% of the animaltest population), or LC_(LO) (lowest dose causing lethality). Almost anydesired effect or endpoint can be characterized in this manner.

The measured dose of a response curve is typically plotted on the X axisand the response is plotted on the Y axis. More typically, the logarithmof the dose is plotted on the X axis, most often generating a sigmoidalcurve with the steepest portion in the middle. The No Observable EffectLevel (NOEL) refers to the lowest experimental dose for which nomeasurable effect is observed, and the threshold dose refers to thefirst point along the graph that indicates a response above zero. As ageneral rule, stronger drugs generate steeper dose response curves. Formany drugs, the desired effects are found at doses slightly greater thanthe threshold dose, often because lower doses are relatively ineffectiveand higher doses lead to undesired side effects. For in vivo generateddose response curves, a curve can be characterized by values such asμg/kg, mg/kg, or g/kg of body-weight, if desired.

For batch comparisons, it can be useful to calculate the coefficient ofvariation (CV) between different dose response curves of differentbatches (e.g., between different batches of AARS protein fragments,antibodies, or other agents), in part because the CV allows comparisonbetween data sets with different units or different means. For instance,in certain exemplary embodiments, two or three or more different batchesof AARS protein fragments or other agents have a CV between them of lessthan about 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or 1% for a 4, 5, 6, 7, or 8 point dose curve. In certain embodiments,the dose response curve is measured in a cell-based assay, and itsreadout relates to an increase or a decrease in a selected non-canonicalactivity of the AARS protein fragment. In certain embodiments, the doseresponse curve is measured in a cell release assay or animal model(e.g., mouse model), and its readout relates to cell death or animaldeath. Other variations will be apparent to persons skilled in the art.

VIII. EXPRESSION AND PURIFICATION SYSTEMS

Embodiments of the present invention include methods and relatedcompositions for expressing and purifying the AARS protein fragments orother polypeptide-based agents of the invention. Such recombinant AARSpolypeptides can be conveniently prepared using standard protocols asdescribed for example in Sambrook, et al., (1989, supra), in particularSections 16 and 17; Ausubel et al., (1994, supra), in particularChapters 10 and 16; and Coligan et al., Current Protocols in ProteinScience (John Wiley & Sons, Inc. 1995-1997), in particular Chapters 1, 5and 6. As one general example, AARS polypeptides may be prepared by aprocedure including one or more of the steps of: (a) preparing aconstruct comprising a polynucleotide sequence that encodes a AARSpolypeptide and that is operably linked to a regulatory element; (b)introducing the construct into a host cell; (c) culturing the host cellto express the AARS polypeptide; and (d) isolating the AARS polypeptidefrom the host cell.

AARS polynucleotides are described elsewhere herein. In order to expressa desired polypeptide, a nucleotide sequence encoding the polypeptide,or a functional equivalent, may be inserted into appropriate expressionvector, i.e., a vector which contains the necessary elements for thetranscription and translation of the inserted coding sequence. Methodswhich are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding a polypeptideof interest and appropriate transcriptional and translational controlelements. These methods include in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. Such techniquesare described in Sambrook et al., Molecular Cloning, A Laboratory Manual(1989), and Ausubel et al., Current Protocols in Molecular Biology(1989).

A variety of expression vector/host systems are known and may beutilized to contain and express polynucleotide sequences. These include,but are not limited to, microorganisms such as bacteria transformed withrecombinant bacteriophage, plasmid, or cosmid DNA expression vectors;yeast transformed with yeast expression vectors; insect cell systemsinfected with virus expression vectors (e.g., baculovirus); plant cellsystems transformed with virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterialexpression vectors (e.g., Ti or pBR322 plasmids); or animal cellsystems, including mammalian cell and more specifically human cellsystems.

The “control elements” or “regulatory sequences” present in anexpression vector are those non-translated regions of thevector—enhancers, promoters, 5′ and 3′ untranslated regions—whichinteract with host cellular proteins to carry out transcription andtranslation. Such elements may vary in their strength and specificity.Depending on the vector system and host utilized, any number of suitabletranscription and translation elements, including constitutive andinducible promoters, may be used. For example, when cloning in bacterialsystems, inducible promoters such as the hybrid lacZ promoter of thePBLUESCRIPT phagemid (Stratagene, La Jolla, Calif) or PSPORT1 plasmid(Gibco BRL, Gaithersburg, Md.) and the like may be used. In mammaliancell systems, promoters from mammalian genes or from mammalian virusesare generally preferred. If it is necessary to generate a cell line thatcontains multiple copies of the sequence encoding a polypeptide, vectorsbased on SV40 or EBV may be advantageously used with an appropriateselectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for the expressed polypeptide. Forexample, when large quantities are needed, vectors which direct highlevel expression of fusion proteins that are readily purified may beused. Such vectors include, but are not limited to, the multifunctionalE. coli cloning and expression vectors such as BLUESCRIPT (Stratagene),in which the sequence encoding the polypeptide of interest may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke & Schuster, J. Biol. Chem.264:5503 5509 (1989)); and the like. pGEX Vectors (Promega, Madison,Wis.) may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. Proteins made in such systems may bedesigned to include heparin, thrombin, or factor XA protease cleavagesites so that the cloned polypeptide of interest can be released fromthe GST moiety at will.

Certain embodiments may employ E. coli-based expression systems (see,e.g., Structural Genomics Consortium et al., Nature Methods. 5:135-146,2008). These and related embodiments may rely partially or totally onligation-independent cloning (LIC) to produce a suitable expressionvector. In specific embodiments, protein expression may be controlled bya T7 RNA polymerase (e.g., pET vector series). These and relatedembodiments may utilize the expression host strain BL21(DE3), a λDE3lysogen of BL21 that supports T7-mediated expression and is deficient inlon and ompT proteases for improved target protein stability. Alsoincluded are expression host strains carrying plasmids encoding tRNAsrarely used in E. coli, such as ROSETTA™ (DE3) and Rosetta 2 (DE3)strains. Cell lysis and sample handling may also be improved usingreagents sold under the trademarks BENZONASE® nuclease and BUGBUSTER®Protein Extraction Reagent. For cell culture, auto-inducing media canimprove the efficiency of many expression systems, includinghigh-throughput expression systems. Media of this type (e.g., OVERNIGHTEXPRESS™ Autoinduction System) gradually elicit protein expressionthrough metabolic shift without the addition of artificial inducingagents such as IPTG. Particular embodiments employ hexahistidine tags(such as those sold under the trademark HIS•TAG® fusions), followed byimmobilized metal affinity chromatography (IMAC) purification, orrelated techniques. In certain aspects, however, clinical grade proteinscan be isolated from E. coli inclusion bodies, without or without theuse of affinity tags (see, e.g., Shimp et al., Protein Expr Purif.50:58-67, 2006). As a further example, certain embodiments may employ acold-shock induced E. coli high-yield production system, becauseover-expression of proteins in Escherichia coli at low temperatureimproves their solubility and stability (see, e.g., Qing et al., NatureBiotechnology. 22:877-882, 2004).

Also included are high-density bacterial fermentation systems. Forexample, high cell density cultivation of Ralstonia eutropha allowsprotein production at cell densities of over 150 g/L, and the expressionof recombinant proteins at titers exceeding 10 g/L.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al., Methods Enzymol. 153:516-544 (1987). Also included arePichia pandoris expression systems (see, e.g., Li et al., NatureBiotechnology. 24, 210-215, 2006; and Hamilton et al., Science,301:1244, 2003). Certain embodiments include yeast systems that areengineered to selectively glycosylate proteins, including yeast thathave humanized N-glycosylation pathways, among others (see, e.g.,Hamilton et al., Science. 313:1441-1443, 2006; Wildt et al., NatureReviews Microbiol. 3:119-28, 2005; and Gerngross et al.,Nature-Biotechnology. 22:1409-1414, 2004; U.S. Pat. Nos. 7,629,163;7,326,681; and 7,029,872). Merely by way of example, recombinant yeastcultures can be grown in Fernbach Flasks or 15 L, SOL, 100 L, and 200 Lfermentors, among others.

In cases where plant expression vectors are used, the expression ofsequences encoding polypeptides may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, EMBO J. 6:307-311 (1987)).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi et al., EMBO J. 3:1671-1680(1984); Broglie et al., Science 224:838-843 (1984); and Winter et al.,Results Probl. Cell Differ. 17:85-105 (1991)). These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews (see, e.g., Hobbs in McGraw Hill,Yearbook of Science and Technology, pp. 191-196 (1992)).

An insect system may also be used to express a polypeptide of interest.For example, in one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia cells. The sequencesencoding the polypeptide may be cloned into a non-essential region ofthe virus, such as the polyhedrin gene, and placed under control of thepolyhedrin promoter. Successful insertion of the polypeptide-encodingsequence will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusiacells in which the polypeptide of interest may be expressed (Engelhardet al., Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227 (1994)). Alsoincluded are baculovirus expression systems, including those thatutilize SF9, SF21, and T. ni cells (see, e.g., Murphy and Piwnica-Worms,Curr Protoc Protein Sci. Chapter 5:Unit5.4, 2001). Insect systems canprovide post-translation modifications that are similar to mammaliansystems.

In mammalian host cells, a number of viral-based expression systems aregenerally available. For example, in cases where an adenovirus is usedas an expression vector, sequences encoding a polypeptide of interestmay be ligated into an adenovirus transcription/translation complexconsisting of the late promoter and tripartite leader sequence.Insertion in a non-essential E1 or E3 region of the viral genome may beused to obtain a viable virus which is capable of expressing thepolypeptide in infected host cells (Logan & Shenk, Proc. Natl. Acad.Sci. U.S.A. 81:3655-3659 (1984)). In addition, transcription enhancers,such as the Rous sarcoma virus (RSV) enhancer, may be used to increaseexpression in mammalian host cells.

Examples of useful mammalian host cell lines include monkey kidney CV1line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidneyline (293 or 293 cells sub-cloned for growth in suspension culture,Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells(BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2). Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al., PNASUSA 77:4216 (1980)); and myeloma cell lines such as NSO and Sp2/0. For areview of certain mammalian host cell lines suitable for antibodyproduction, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol.248 (B. K. C Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 255-268.Certain preferred mammalian cell expression systems include CHO andHEK293-cell based expression systems. Mammalian expression systems canutilize attached cell lines, for example, in T-flasks, roller bottles,or cell factories, or suspension cultures, for example, in 1 L and 5 Lspinners, 5 L, 14 L, 40 L, 100 L and 200 L stir tank bioreactors, or20/50 L and 100/200 L WAVE bioreactors, among others known in the art.

Also included is cell-free expression of proteins. These and relatedembodiments typically utilize purified RNA polymerase, ribosomes, tRNAand ribonucleotides; these reagents may be produced by extraction fromcells or from a cell-based expression system.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding a polypeptide of interest. Suchsignals include the ATG initiation codon and adjacent sequences. Incases where sequences encoding the polypeptide, its initiation codon,and upstream sequences are inserted into the appropriate expressionvector, no additional transcriptional or translational control signalsmay be needed. However, in cases where only coding sequence, or aportion thereof, is inserted, exogenous translational control signalsincluding the ATG initiation codon should be provided. Furthermore, theinitiation codon should be in the correct reading frame to ensuretranslation of the entire insert. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers which are appropriate for the particular cell system which isused, such as those described in the literature (Scharf et al., ResultsProbl. Cell Differ. 20:125-162 (1994)).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, post-translationalmodifications such as acetylation, carboxylation, glycosylation,phosphorylation, lipidation, and acylation. Post-translationalprocessing which cleaves a “prepro” form of the protein may also be usedto facilitate correct insertion, folding and/or function. Different hostcells such as yeast, CHO, HeLa, MDCK, HEK293, and W138, in addition tobacterial cells, which have or even lack specific cellular machinery andcharacteristic mechanisms for such post-translational activities, may bechosen to ensure the correct modification and processing of the foreignprotein.

For long-term, high-yield production of recombinant proteins, stableexpression is generally preferred. For example, cell lines which stablyexpress a polynucleotide of interest may be transformed using expressionvectors which may contain viral origins of replication and/or endogenousexpression elements and a selectable marker gene on the same or on aseparate vector. Following the introduction of the vector, cells may beallowed to grow for about 1-2 days in an enriched media before they areswitched to selective media. The purpose of the selectable marker is toconfer resistance to selection, and its presence allows growth andrecovery of cells which successfully express the introduced sequences.Resistant clones of stably transformed cells may be proliferated usingtissue culture techniques appropriate to the cell type. Transientproduction, such as by transient transfection or infection, can also beemployed. Exemplary mammalian expression systems that are suitable fortransient production include HEK293 and CHO-based systems.

Any number of selection systems may be used to recover transformed ortransduced cell lines. These include, but are not limited to, the herpessimplex virus thymidine kinase (Wigler et al., Cell 11:223-232 (1977))and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817-823(1990)) genes which can be employed in tk- or aprt-cells, respectively.Also, antimetabolite, antibiotic or herbicide resistance can be used asthe basis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler et al., Proc. Natl. Acad. Sci. U.S.A. 77:3567-70(1980)); npt, which confers resistance to the aminoglycosides, neomycinand G-418 (Colbere-Garapin et al., J. Mol. Biol. 150:1-14 (1981)); andals or pat, which confer resistance to chlorsulfuron and phosphinotricinacetyltransferase, respectively (Murry, supra). Additional selectablegenes have been described, for example, trpB, which allows cells toutilize indole in place of tryptophan, or hisD, which allows cells toutilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl.Acad. Sci. U.S.A. 85:8047-51 (1988)). The use of visible markers hasgained popularity with such markers as green fluorescent protein (GFP)and other fluorescent proteins (e.g., RFP, YFP), anthocyanins,β-glucuronidase and its substrate GUS, and luciferase and its substrateluciferin, being widely used not only to identify transformants, butalso to quantify the amount of transient or stable protein expressionattributable to a specific vector system (see, e.g., Rhodes et al.,Methods Mol. Biol. 55:121-131 (1995)).

Embodiments of the present invention also include high-throughputprotein production systems, or micro-production systems. Certain aspectsmay utilize, for example, hexa-histidine fusion tags for proteinexpression and purification on metal chelate-modified slide surfaces orMagneHis Ni-Particles (see, e.g., Kwon et al., BMC Biotechnol. 9:72,2009; and Lin et al., Methods Mol Biol. 498:129-41, 2009)). Alsoincluded are high-throughput cell-free protein expression systems (see,e.g., Sitaraman et al., Methods Mol Biol. 498:229-44, 2009). These andrelated embodiments can be used, for example, to generate microarrays ofAARS protein fragment(s), which can then be used for screening librariesto identify agents that interact with the AARS protein fragment(s).

A variety of protocols for detecting and measuring the expression ofpolynucleotide-encoded products, using binding agents or antibodies suchas polyclonal or monoclonal antibodies specific for the product, areknown in the art. Examples include enzyme-linked immunosorbent assay(ELISA), western immunoblots, radioimmunoassays (RIA), and fluorescenceactivated cell sorting (FACS). These and other assays are described,among other places, in Hampton et al., Serological Methods, a LaboratoryManual (1990) and Maddox et al., J. Exp. Med. 158:1211-1216 (1983).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides include oligolabeling,nick translation, end-labeling or PCR amplification using a labelednucleotide. Alternatively, the sequences, or any portions thereof may becloned into a vector for the production of an mRNA probe. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by addition of an appropriate RNApolymerase such as T7, T3, or SP6 and labeled nucleotides. Theseprocedures may be conducted using a variety of commercially availablekits. Suitable reporter molecules or labels, which may be used includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with a polynucleotide sequence of interest may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. Certain specific embodiments utilizeserum free cell expression systems. Examples include HEK293 cells andCHO cells that can grown on serum free medium (see, e.g., Rosser et al.,Protein Expr. Purif. 40:237-43, 2005; and U.S. Pat. No. 6,210,922).

The protein produced by a recombinant cell may be secreted or containedintracellularly depending on the sequence and/or the vector used. Aswill be understood by those of skill in the art, expression vectorscontaining polynucleotides of the invention may be designed to containsignal sequences which direct secretion of the encoded polypeptidethrough a prokaryotic or eukaryotic cell membrane. Other recombinantconstructions may be used to join sequences encoding a polypeptide ofinterest to nucleotide sequence encoding a polypeptide domain which willfacilitate purification and/or detection of soluble proteins. Examplesof such domains include cleavable and non-cleavable affinitypurification and epitope tags such as avidin, FLAG tags, poly-histidinetags (e.g., 6×His), cMyc tags, VS-tags, glutathione 5-transferase (GST)tags, and others.

The protein produced by a recombinant cell can be purified andcharacterized according to a variety of techniques known in the art.Exemplary systems for performing protein purification and analyzingprotein purity include fast protein liquid chromatography (FPLC) (e.g.,AKTA and Bio-Rad FPLC systems), high-pressure liquid chromatography(HPLC) (e.g., Beckman and Waters HPLC). Exemplary chemistries forpurification include ion exchange chromatography (e.g., Q, S), sizeexclusion chromatography, salt gradients, affinity purification (e.g.,Ni, Co, FLAG, maltose, glutathione, protein A/G), gel filtration,reverse-phase, ceramic HYPERD® ion exchange chromatography, andhydrophobic interaction columns (HIC), among others known in the art.Also included are analytical methods such as SDS-PAGE (e.g., coomassie,silver stain), immunoblot, Bradford, and ELISA, which may be utilizedduring any step of the production or purification process, typically tomeasure the purity of the protein composition.

Also included are methods of concentrating AARS protein fragments, andcomposition comprising concentrated soluble proteins. In differentaspects such concentrated solutions of AARS polypeptides may compriseproteins at a concentration of about 5 mg/mL; or about 8 mg/mL; or about10 mg/mL; about 15 mg/mL; or about 20 mg/mL.

In one aspect such compositions may be substantially monodisperse,meaning that the AARS polypeptide compositions exist primarily (i.e. atleast about 90%, or greater) in one apparent molecular weight form whenassessed for example, by size exclusion chromatography, dynamic lightscattering, or analytical ultracentrifugation.

In another aspect, such compositions have a purity (on a protein basis)of at least about 90%, or in some aspects at least about 95% purity, orin some embodiments, at least 98% purity. Purity may be determined viaany routine analytical method as known in the art.

In another aspect, such compositions have a high molecular weightaggregate content of less than about 10%, compared to the total amountof protein present, or in some embodiments such compositions have a highmolecular weight aggregate content of less than about 5%, or in someaspects such compositions have a high molecular weight aggregate contentof less than about 3%, or in some embodiments a high molecular weightaggregate content of less than about 1%. High molecular weight aggregatecontent may be determined via a variety of analytical techniquesincluding for example, by size exclusion chromatography, dynamic lightscattering, or analytical ultracentrifugation.

In certain embodiments, as noted herein, the AARS polypeptidecompositions have an endotoxin content of less than about 10 EU/mg ofAARS polypeptide, or less than about 5 EU/mg of AARS polypeptide, lessthan about 3 EU/mg of AARS polypeptide, or less than about 1 EU/mg ofAARS polypeptide.

Examples of concentration approaches contemplated herein includelyophilization, which is typically employed when the solution containsfew soluble components other than the protein of interest.Lyophilization is often performed after HPLC run, and can remove most orall volatile components from the mixture. Also included areultrafiltration techniques, which typically employ one or more selectivepermeable membranes to concentrate a protein solution. The membraneallows water and small molecules to pass through and retains theprotein; the solution can be forced against the membrane by mechanicalpump, gas pressure, or centrifugation, among other techniques.

In certain embodiments, the reagents, AARS protein fragments, or relatedagents (e.g., antibodies) have a purity of at least about 90%, asmeasured according to routine techniques in the art. In certainembodiments, such as diagnostic compositions or certain therapeuticcompositions, the AARS compositions of the present invention have apurity of at least about 95%. In specific embodiments, such astherapeutic or pharmaceutical compositions, the AARS compositions of thepresent invention have a purity of at least about 97% or 98% or 99%. Inother embodiments, such as when being used as reference or researchreagents, AARS protein fragments can be of lesser purity, and may have apurity of at least about 50%, 60%, 70%, or 80%. Purity can be measuredoverall or in relation to selected components, such as other proteins,e.g., purity on a protein basis.

Purified AARS protein fragments can also be characterized according totheir biological characteristics. Examples include binding affinity orbinding kinetics to a selected ligand (e.g., a cellular binding partnerof the AARS protein fragment such as a cell-surface receptor or anextracellular domain thereof), and the presence or levels of one or morecanonical or non-canonical biological activity, as described herein.Binding affinity and binding kinetics can be measured according to avariety of techniques known in the art, such as Biacore® and relatedtechnologies that utilize surface plasmon resonance (SPR), an opticalphenomenon that enables detection of unlabeled interactants in realtime. SPR-based biosensors can be used in determination of activeconcentration, screening and characterization in terms of both affinityand kinetics. The presence or levels of one or more canonical ornon-canonical biological activities can be measured according tocell-based assays, including those that utilize a cellular bindingpartner (e.g., cell-surface receptor) of a selected AARS proteinfragment, which is functionally coupled to a readout or indicator, suchas a fluorescent or luminescent indicator of a non-canonical biologicalactivity, as described herein.

In certain embodiments, as noted above, the AARS polypeptidecompositions are about substantially endotoxin free, including, forexample, about 95% endotoxin free, preferably about 99% endotoxin free,and more preferably about 99.99% endotoxin free. The presence ofendotoxins can be detected according to routine techniques in the art,as described herein. In specific embodiments, the AARS compositions aremade from a eukaryotic cell such as a mammalian or human cell insubstantially serum free media.

In certain embodiments, the AARS polypeptide compositions comprise lessthan about 10% wt/wt high molecular weight aggregates, or less thanabout 5% wt/wt high molecular weight aggregates, or less than about 2%wt/wt high molecular weight aggregates, or less than about or less thanabout 1% wt/wt high molecular weight aggregates.

Also included are protein-based analytical assays and methods, which canbe used to assess, for example, protein purity, size, solubility, anddegree of aggregation, among other characteristics. Protein purity canbe assessed a number of ways. For instance, purity can be assessed basedon primary structure, higher order structure, size, charge,hydrophobicity, and glycosylation. Examples of methods for assessingprimary structure include N- and C-terminal sequencing andpeptide-mapping (see, e.g., Allen et al., Biologicals. 24:255-275,1996)). Examples of methods for assessing higher order structure includecircular dichroism (see, e.g., Kelly et al., Biochim Biophys Acta.1751:119-139, 2005), fluorescent spectroscopy (see, e.g., Meagher etal., J. Biol. Chem. 273:23283-89, 1998), FT-IR, amide hydrogen-deuteriumexchange kinetics, differential scanning calorimetry, NMR spectroscopy,immunoreactivity with conformationally sensitive antibodies. Higherorder structure can also be assessed as a function of a variety ofparameters such as pH, temperature, or added salts. Examples of methodsfor assessing protein characteristics such as size include analyticalultracentrifugation and size exclusion HPLC (SEC-HPLC), and exemplarymethods for measuring charge include ion-exchange chromatography andisolectric focusing. Hydrophobicity can be assessed, for example, byreverse-phase HPLC and hydrophobic interaction chromatography HPLC.Glycosylation can affect pharmacokinetics (e.g., clearance),conformation or stability, receptor binding, and protein function, andcan be assessed, for example, by mass spectrometry and nuclear magneticresonance (NMR) spectroscopy.

As noted above, certain embodiments include the use of SEC-HPLC toassess protein characteristics such as purity, size (e.g., sizehomogeneity) or degree of aggregation, and/or to purify proteins, amongother uses. SEC, also including gel-filtration chromatography (GFC) andgel-permeation chromatography (GPC), refers to a chromatographic methodin which molecules in solution are separated in a porous material basedon their size, or more specifically their hydrodynamic volume, diffusioncoefficient, and/or surface properties. The process is generally used toseparate biological molecules, and to determine molecular weights andmolecular weight distributions of polymers. Typically, a biological orprotein sample (such as a protein extract produced according to theprotein expression methods provided herein and known in the art) isloaded into a selected size-exclusion column with a defined stationaryphase (the porous material), preferably a phase that does not interactwith the proteins in the sample. In certain aspects, the stationaryphase is composed of inert particles packed into a densethree-dimensional matrix within a glass or steel column. The mobilephase can be pure water, an aqueous buffer, an organic solvent, or amixture thereof. The stationary-phase particles typically have smallpores and/or channels which only allow molecules below a certain size toenter. Large particles are therefore excluded from these pores andchannels, and their limited interaction with the stationary phase leadsthem to elute as a “totally-excluded” peak at the beginning of theexperiment. Smaller molecules, which can fit into the pores, are removedfrom the flowing mobile phase, and the time they spend immobilized inthe stationary-phase pores depends, in part, on how far into the poresthey penetrate. Their removal from the mobile phase flow causes them totake longer to elute from the column and results in a separation betweenthe particles based on differences in their size. A given size exclusioncolumn has a range of molecular weights that can be separated. Overall,molecules larger than the upper limit will not be trapped by thestationary phase, molecules smaller than the lower limit will completelyenter the solid phase and elute as a single band, and molecules withinthe range will elute at different rates, defined by their propertiessuch as hydrodynamic volume. For examples of these methods in practicewith pharmaceutical proteins, see Bruner et al., Journal ofPharmaceutical and Biomedical Analysis. 15: 1929-1935, 1997.

Protein purity for clinical applications is also discussed, for example,by Anicetti et al. (Trends in Biotechnology. 7:342-349, 1989). Morerecent techniques for analyzing protein purity include, withoutlimitation, the LabChip GXII, an automated platform for rapid analysisof proteins and nucleic acids, which provides high throughput analysisof titer, sizing, and purity analysis of proteins. In certainnon-limiting embodiments, clinical grade proteins such as proteinfragments and antibodies can be obtained by utilizing a combination ofchromatographic materials in at least two orthogonal steps, among othermethods (see, e.g., Therapeutic Proteins: Methods and Protocols. Vol.308, Eds., Smales and James, Humana Press Inc., 2005). Typically,protein agents (e.g., AARS protein fragments, antibodies, bindingagents) and other agents (e.g., antisense, RNAi, small molecules) aresubstantially endotoxin-free, as measured according to techniques knownin the art and described herein.

Protein solubility assays are also included. Such assays can beutilized, for example, to determine optimal growth and purificationconditions for recombinant production, to optimize the choice ofbuffer(s), and to optimize the choice of AARS protein fragments orvariants thereof. Solubility or aggregation can be evaluated accordingto a variety of parameters, including temperature, pH, salts, and thepresence or absence of other additives. Examples of solubility screeningassays include, without limitation, microplate-based methods ofmeasuring protein solubility using turbidity or other measure as an endpoint, high-throughput assays for analysis of the solubility of purifiedrecombinant proteins (see, e.g., Stenvall et al., Biochim Biophys Acta.1752:6-10, 2005), assays that use structural complementation of agenetic marker protein to monitor and measure protein folding andsolubility in vivo (see, e.g., Wigley et al., Nature Biotechnology.19:131-136, 2001), and electrochemical screening of recombinant proteinsolubility in Escherichia coli using scanning electrochemical microscopy(SECM) (see, e.g., Nagamine et al., Biotechnology and Bioengineering.96:1008-1013, 2006), among others. AARS protein fragments with increasedsolubility (or reduced aggregation) can be identified or selected foraccording to routine techniques in the art, including simple in vivoassays for protein solubility (see, e.g., Maxwell et al., Protein Sci.8:1908-11, 1999).

Protein solubility and aggregation can also be measured by dynamic lightscattering techniques. Aggregation is a general term that encompassesseveral types of interactions or characteristics, includingsoluble/insoluble, covalent/noncovalent, reversible/irreversible, andnative/denatured interactions and characteristics. For proteintherapeutics, the presence of aggregates is typically consideredundesirable because of the concern that aggregates may cause animmunogenic reaction (e.g., small aggregates), or may cause adverseevents on administration (e.g., particulates). Dynamic light scatteringrefers to a technique that can be used to determine the sizedistribution profile of small particles in suspension or polymers suchas proteins in solution. This technique, also referred to as photoncorrelation spectroscopy (PCS) or quasi-elastic light scattering (QELS),uses scattered light to measure the rate of diffusion of the proteinparticles. Fluctuations of the scattering intensity can be observed dueto the Brownian motion of the molecules and particles in solution. Thismotion data can be conventionally processed to derive a sizedistribution for the sample, wherein the size is given by the Stokesradius or hydrodynamic radius of the protein particle. The hydrodynamicsize depends on both mass and shape (conformation). Dynamic scatteringcan detect the presence of very small amounts of aggregated protein(<0.01% by weight), even in samples that contain a large range ofmasses. It can also be used to compare the stability of differentformulations, including, for example, applications that rely onreal-time monitoring of changes at elevated temperatures. Accordingly,certain embodiments include the use of dynamic light scattering toanalyze the solubility and/or presence of aggregates in a sample thatcontains an AARS protein fragment, antibody, or other agent of theinvention.

IX. DIAGNOSTIC METHODS AND COMPOSITIONS

AARS agents such as AARS protein fragments, AARS polynucleotides, andantibodies and other binding agents described herein can be used indiagnostic assays and diagnostic compositions. Included are biochemical,histological, and cell-based methods and compositions, among others.

These and related embodiments include the detection of the AARSpolynucleotide sequence(s) or corresponding AARS polypeptide sequence(s)or portions thereof of one or more newly identified AARS proteinfragments, also referred to as AARS polypeptides. For instance, certainaspects include detection of the AARS polynucleotide sequence(s) orcorresponding polypeptide sequence(s) or portions thereof of one or morenewly identified AARS splice variants, and/or one or more splicejunctions of those splice variants. In certain embodiments, thepolynucleotide or corresponding polypeptide sequence(s) of at least oneof the splice junctions is unique to that particular AARS splicevariant.

Also included is the direct detection of AARS protein fragments,including splice variants, proteolytic fragments, and others. In certainembodiments, the presence or levels of one or more newly identified AARSprotein fragments associate or correlate with one or more cellular typesor cellular states. Hence, the presence or levels of an AARS polypeptideor polynucleotide can be used to distinguish between different cellulartypes or different cellular states. The presence or levels of AARSprotein fragments or their related polynucleotides can be detectedaccording to polynucleotide and/or polypeptide-based diagnostictechniques, as described herein and known in the art.

Certain aspects can employ the AARS protein fragments, antibody, or AARSpolynucleotides as part of a companion diagnostic method, typically toassess whether a subject or population subjects will respond favorablyto a specific medical treatment. For instance, a given AARS therapeuticagent (e.g., protein fragment, antisense, RNAi, antibody, binding agent)could be identified as suitable for a subject or certain populations ofsubjects based on whether the subject(s) have one or more selectedbiomarkers for a given disease or condition. Examples of biomarkersinclude serum/tissue markers as well as markers that can be identifiedby medical imaging techniques. In certain embodiments, anaturally-occurring AARS protein fragment (or its correspondingpolynucleotide) may itself provide a serum and/or tissue biomarker thatcan be utilized to measure drug outcome or assess the desirability ofdrug use in a specific subject or a specific population of subjects. Incertain aspects, the identification of an AARS polypeptide orpolynucleotide reference sequence may include characterizing thedifferential expression of that sequence, whether in a selected subject,selected tissue, or otherwise, as described herein and known in the art.

Certain of the methods provided herein rely on the differentialexpression of an AARS polypeptide or polynucleotide to characterize thecondition or state of a cell, tissue, or subject, and to distinguish itfrom another cell, tissue, or subject. Non-limiting examples includemethods of detecting the presence or levels of an AARS polypeptide orpolynucleotide in a biological sample to distinguish between cells ortissues of different species, cells of different tissues or organs,cellular developmental states such as neonatal and adult, cellulardifferentiation states, conditions such as healthy, diseased andtreated, intracellular and extracellular fractions, in addition toprimary cell cultures and other cell cultures, such as immortalized cellcultures.

Differential expression includes a statistically significant differencein one or more gene expression levels of an AARS polynucleotide orpolypeptide reference sequence compared to the expression levels of thesame sequence in an appropriate control. The statistically significantdifference may relate to either an increase or a decrease in expressionlevels, as measured by RNA levels, protein levels, protein function, orany other relevant measure of gene expression such as those describedherein. Also included is a comparison between an AARS polynucleotide orpolypeptide of the invention and a full-length or wild-type cytosolic ormitochondrial AARS sequence, typically of the same or correspondingtype. Differential expression can be detected by a variety of techniquesin the art and described herein, including polynucleotide andpolypeptide based techniques, such as real-time PCR, subtractivehybridization, polynucleotide and polypeptide arrays, and others.

A result is typically referred to as statistically significant if it isunlikely to have occurred by chance. The significance level of a test orresult relates traditionally to a frequentist statistical hypothesistesting concept. In simple cases, statistical significance may bedefined as the probability of making a decision to reject the nullhypothesis when the null hypothesis is actually true (a decision knownas a Type I error, or “false positive determination”). This decision isoften made using the p-value: if the p-value is less than thesignificance level, then the null hypothesis is rejected. The smallerthe p-value, the more significant the result. Bayes factors may also beutilized to determine statistical significance (see, e.g., Goodman S.,Ann Intern Med 130:1005-13, 1999).

In more complicated, but practically important cases, the significancelevel of a test or result may reflect an analysis in which theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true is no more than the stated probability.This type of analysis allows for those applications in which theprobability of deciding to reject may be much smaller than thesignificance level for some sets of assumptions encompassed within thenull hypothesis.

In certain exemplary embodiments, statistically significant differentialexpression may include situations wherein the expression level of agiven AARS sequence provides at least about a 1.2×, 1.3×, 1.4×, 1.5×,1.6×, 1.7×, 1.8×, 1.9×, 2.0×, 2.2×, 2.4×, 2.6×, 2.8×, 3.0×, 4.0×, 5.0×,6.0×, 7.0×, 8.0×, 9.0×, 10.0×, 15.0×, 20.0×, 50.0×, 100.0×, or greaterdifference in expression (i.e., differential expression that may behigher or lower expression) in a suspected biological sample as comparedto an appropriate control, including all integers and decimal points inbetween (e.g., 1.24×, 1.25×, 2.1×, 2.5×, 60.0×, 75.0×, etc.). In certainembodiments, statistically significant differential expression mayinclude situations wherein the expression level of a given AARS sequenceprovides at least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000 percent (%) or greater difference inexpression (i.e., differential expression that may be higher or lower)in a suspected biological sample as compared to an appropriate control,including all integers and decimal points in between.

As an additional example, differential expression may also be determinedby performing Z-testing, i.e., calculating an absolute Z score, asdescribed herein and known in the art (see Example 1). Z-testing istypically utilized to identify significant differences between a samplemean and a population mean. For example, as compared to a standardnormal table (e.g., a control tissue), at a 95% confidence interval(i.e., at the 5% significance level), a Z-score with an absolute valuegreater than 1.96 indicates non-randomness. For a 99% confidenceinterval, if the absolute Z is greater than 2.58, it means that p<0.01,and the difference is even more significant—the null hypothesis can berejected with greater confidence. In these and related embodiments, anabsolute Z-score of 1.96, 2, 2.58, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or more, including all decimal points inbetween (e.g., 10.1, 10.6, 11.2, etc.), may provide a strong measure ofstatistical significance. In certain embodiments, an absolute Z-score ofgreater than 6 may provide exceptionally high statistical significance.

Substantial similarly relates generally to the lack of a statisticallysignificant difference in the expression levels between the biologicalsample and the reference control. Examples of substantially similarexpression levels may include situations wherein the expression level ofa given SSCIGS provides less than about a 0.05×, 0.1×, 0.2×, 0.3×, 0.4×,0.5×, 0.6×, 0.7×, 0.8×, 0.9×, 1.0×, 1.1×, 1.2×, 1.3×, or 1.4× differencein expression (i.e., differential expression that may be higher or lowerexpression) in a suspected biological sample as compared to a referencesample, including all decimal points in between (e.g., 0.15×, 0.25×,0.35×, etc.). In certain embodiments, differential expression mayinclude situations wherein the expression level of a given AARS sequenceprovides less than about 0.25. 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50 percent (%) difference inexpression (i.e., differential expression that may be higher or lower)in a suspected biological sample as compared to a reference sample,including all decimal points in between.

In certain embodiments, such as when using an Affymetrix Microarray tomeasure the expression levels of an AARS polynucleotide or polypeptidereference sequence, differential expression may also be determined bythe mean expression value summarized by Affymetrix Microarray Suite 5software (Affymetrix, Santa Clara, Calif.), or other similar software,typically with a scaled mean expression value of 1000.

Embodiments of the present invention include methods of detecting thepresence or levels of an AARS polynucleotide or polypeptide referencesequence or a portion thereof to distinguish between cells or tissues orother biological sample of a different organism or species, wherein thepresence or levels of that sequence associates with a selected organismor species. General examples include methods of distinguishing betweenhumans and any combination of bacteria, fungi, plants, and othernon-human animals. Included within animals are methods of distinguishingbetween humans and any combination of vertebrates and invertebrates,including vertebrates such as fish, amphibians, reptiles, birds, andnon-human mammals, and invertebrates such as insects, mollusks,crustaceans, and corals. Included within non-human mammals are methodsof distinguishing between humans and any combination of non-humanmammals from the Order Afrosoricida, Macroscelidea, Tubulidentata,Hyracoidea, Proboscidea, Sirenia, Cingulata, Pilosa, Scandentia,Dermoptera, Primates, Rodentia, Lagomorpha, Erinaceomorpha,Soricomorpha, Chiroptera, Pholidota, Cetacea, Carnivora, Perissodactyla,or Artiodactyla. Included within the Primate Order are monkeys, apes,gorillas, and chimpanzees, among others known in the art. Accordingly,the presence or levels of an AARS polynucleotide or polypeptidereference sequence or variant, as described herein, may be used toidentify the source of a given biological sample, such as a cell,tissue, or organ, by distinguishing between any combination of theseorganisms, or by distinguishing between humans and any one or more ofthese organisms, such as a panel of organisms. In certain embodiments,the source of a given biological sample may also be determined bycomparing the presence or levels of an AARS sequence or a portionthereof to a pre-determined value.

Embodiments of the present invention include methods of detecting thepresence or levels of an AARS polynucleotide or polypeptide referencesequence or a portion thereof to distinguish between cells or otherbiological samples that originate from different tissues or organs.Non-limiting examples include methods of distinguishing between a cellor other biological sample that originates from any combination of skin(e.g., dermis, epidermis, subcutaneous layer), hair follicles, nervoussystem (e.g., brain, spinal cord, peripheral nerves), auditory system orbalance organs (e.g., inner ear, middle ear, outer ear), respiratorysystem (e.g., nose, trachea, lungs), gastroesophogeal tissues, thegastrointestinal system (e.g., mouth, esophagus, stomach, smallintestines, large intestines, rectum), vascular system (e.g., heart,blood vessels and arteries), liver, gallbladder, lymphatic/immune system(e.g., lymph nodes, lymphoid follicles, spleen, thymus, bone marrow),uro-genital system (e.g., kidneys, ureter, bladder, urethra, cervix,Fallopian tubes, ovaries, uterus, vulva, prostate, bulbourethral glands,epididymis, prostate, seminal vesicles, testicles), musculoskeletalsystem (e.g., skeletal muscles, smooth muscles, bone, cartilage,tendons, ligaments), adipose tissue, mammary tissue, and the endocrinesystem (e.g., hypothalamus, pituitary, thyroid, pancreas, adrenalglands). Hence, based on the association of an AARS polynucleotide orpolypeptide sequence as described herein, these methods may be used toidentify or characterize the tissue or organ from which a cell or otherbiological sample is derived.

Embodiments of the present invention include methods of detecting thepresence or levels of an AARS polynucleotide or polypeptide referencesequence or a portion thereof to distinguish between or characterize thedevelopmental or differentiation state of the cell. Also included aremethods of differentiating between germ cells, stem cells, and somaticcells. Examples of developmental states include neonatal and adult.Examples of cellular differentiation states include all of the discreetand identifiable stages between a totipotent cell, a pluripotent cell, amultipotent progenitor stem cell and a mature, fully differentiatedcell.

A totipotent cell has total potential, typically arises during sexualand asexual reproduction, and includes and spores and zygotes, though incertain instances cells can dedifferentiate and regain totipotency. Apluripotent cell includes a stem cell that has the potential todifferentiate into any of the three germ layers, including the endoderm(interior stomach lining, gastrointestinal tract, the lungs), themesoderm (muscle, bone, blood, urogenital), and the ectoderm (epidermaltissues and nervous system). Multipotent progenitor cells are typicallycapable of differentiating into a limited number of tissue types.Examples of multipotent cells include, without limitation, hematopoieticstem cells (adult stem cells) from the bone marrow that give rise toimmune cells such as red blood cells, white blood cells, and platelets,mesenchymal stem cells (adult stem cells) from the bone marrow that giverise to stromal cells, fat cells, and various types of bone cells,epithelial stem cells (progenitor cells) that give rise to the varioustypes of skin cells, and muscle satellite cells (progenitor cells) thatcontribute to differentiated muscle tissue. Accordingly, the presence orlevels of particular AARS polynucleotide or polypeptide sequence (e.g.,splice junction of an AARS splice variant, AARS proteolytic fragment),can be used to distinguish between or characterize the above-notedcellular differentiation states, as compared to a control or apredetermined level.

Embodiments of the present invention include methods of detecting thepresence or levels of an AARS polynucleotide or polypeptide referencesequence to characterize or diagnose the condition or a cell, tissue,organ, or subject, in which that condition may be characterized ashealthy, diseased, at risk for being diseased, or treated. For suchdiagnostic purposes, the term “diagnostic” or “diagnosed” includesidentifying the presence or nature of a pathologic condition,characterizing the risk of developing such a condition, and/or measuringthe change (or no change) of a pathologic condition in response totherapy. Diagnostic methods may differ in their sensitivity andspecificity. In certain embodiments, the “sensitivity” of a diagnosticassay refers to the percentage of diseased cells, tissues or subjectswhich test positive (percent of “true positives”). Diseased cells,tissues or subjects not detected by the assay are typically referred toas “false negatives.” Cells, tissues or subjects that are not diseasedand which test negative in the assay may be termed “true negatives.” Incertain embodiments, the “specificity” of a diagnostic assay may bedefined as one (1) minus the false positive rate, where the “falsepositive” rate is defined as the proportion of those samples or subjectswithout the disease and which test positive. While a particulardiagnostic method may not provide a definitive diagnosis of a condition,it suffices if the method provides a positive indication that aids indiagnosis.

In certain instances, the presence or risk of developing a pathologiccondition can be diagnosed by comparing the presence or levels of one ormore selected AARS polynucleotide or polypeptide reference sequences orportions thereof that correlate with the condition, whether by increasedor decreased levels, as compared to a suitable control. A “suitablecontrol” or “appropriate control” includes a value, level, feature,characteristic, or property determined in a cell or other biologicalsample of a tissue or organism, e.g., a control or normal cell, tissueor organism, exhibiting, for example, normal traits, such as the absenceof the condition. In certain embodiments, a “suitable control” or“appropriate control” is a predefined value, level, feature,characteristic, or property. Other suitable controls will be apparent topersons skilled in the art. Examples of diseases and conditions aredescribed elsewhere herein.

Embodiments of the present invention include AARS polynucleotide ornucleic acid-based detection techniques, which offer certain advantagesdue to sensitivity of detection. Hence, certain embodiments relate tothe use or detection of AARS polynucleotides as part of a diagnosticmethod or assay. The presence and/or levels of AARS polynucleotides maybe measured by any method known in the art, including hybridizationassays such as Northern blot, quantitative or qualitative polymerasechain reaction (PCR), quantitative or qualitative reverse transcriptasePCR (RT-PCR), microarray, dot or slot blots, or in situ hybridizationsuch as fluorescent in situ hybridization (FISH), among others. Certainof these methods are described in greater detail below.

AARS polynucleotides such as DNA and RNA can be collected and/orgenerated from blood, biological fluids, tissues, organs, cell lines, orother relevant sample using techniques known in the art, such as thosedescribed in Kingston. (2002 Current Protocols in Molecular Biology,Greene Publ. Assoc. Inc. & John Wiley & Sons, Inc., NY, N.Y. (see, e.g.,as described by Nelson et al. Proc Natl Acad Sci USA, 99: 11890-11895,2002) and elsewhere. Further, a variety of commercially available kitsfor constructing RNA are useful for making the RNA to be used in thepresent invention. RNA may be constructed from organs/tissues/cellsprocured from normal healthy subjects; however, this invention alsocontemplates construction of RNA from diseased subjects. Certainembodiments contemplate using any type of organ from any type of subjector animal. For test samples RNA may be procured from an individual(e.g., any animal, including mammals) with or without visible diseaseand from tissue samples, biological fluids (e.g., whole blood) or thelike.

In certain embodiments, amplification or construction of cDNA sequencesmay be helpful to increase detection capabilities. The instantdisclosure, as well as the art, provides the requisite level of detailto perform such tasks. In one exemplary embodiment, whole blood is usedas the source of RNA and accordingly, RNA stabilizing reagents areoptionally used, such as PAX tubes, as described, for example, in Thachet al., J. Immunol. Methods. December 283(1-2):269-279, 2003 and Chai etal., J. Clin. Lab Anal. 19(5):182-188, 2005 (both of which areincorporated by reference). Complementary DNA (cDNA) libraries can begenerated using techniques known in the art, such as those described inAusubel et al. (2001 Current Protocols in Molecular Biology, GreenePubl. Assoc. Inc. & John Wiley & Sons, Inc., NY, N.Y.); Sambrook et al.(1989 Molecular Cloning, Second Ed., Cold Spring Harbor Laboratory,Plainview, N.Y.); Maniatis et al. (1982 Molecular Cloning, Cold SpringHarbor Laboratory, Plainview, N.Y.) and elsewhere. Further, a variety ofcommercially available kits for constructing cDNA libraries are usefulfor making the cDNA libraries of the present invention. Libraries can beconstructed from organs/tissues/cells procured from normal, healthysubjects.

Certain embodiments may employ hybridization methods for detecting AARSpolynucleotide sequences. Methods for conducting polynucleotidehybridization assays have been well developed in the art. Hybridizationassay procedures and conditions will vary depending on the applicationand are selected in accordance with the general binding methods knownincluding those referred to in: Maniatis et al. Molecular Cloning: ALaboratory Manual (2nd Ed. Cold Spring Harbor, N.Y., 1989); Berger andKimmel Methods in Enzymology, Vol. 152, Guide to Molecular CloningTechniques (Academic Press, Inc., San Diego, Calif, 1987); Young andDavis, PNAS. 80: 1194 (1983). Methods and apparatus for carrying outrepeated and controlled hybridization reactions have been described inU.S. Pat. Nos. 5,871,928, 5,874,219, 6,045,996 and 6,386,749, 6,391,623each of which are incorporated herein by reference

Certain embodiments may employ nucleic acid amplification methods fordetecting AARS polynucleotide sequences. The term “amplification” or“nucleic acid amplification” refers to the production of multiple copiesof a target nucleic acid that contains at least a portion of theintended specific target nucleic acid sequence. The multiple copies maybe referred to as amplicons or amplification products. In certainembodiments, the amplified target contains less than the complete targetgene sequence (introns and exons) or an expressed target gene sequence(spliced transcript of exons and flanking untranslated sequences). Forexample, specific amplicons may be produced by amplifying a portion ofthe target polynucleotide by using amplification primers that hybridizeto, and initiate polymerization from, internal positions of the targetpolynucleotide. Preferably, the amplified portion contains a detectabletarget sequence that may be detected using any of a variety ofwell-known methods.

“Selective amplification” or “specific amplification,” as used herein,refers to the amplification of a target nucleic acid sequence accordingto the present invention wherein detectable amplification of the targetsequence is substantially limited to amplification of target sequencecontributed by a nucleic acid sample of interest that is being testedand is not contributed by target nucleic acid sequence contributed bysome other sample source, e.g., contamination present in reagents usedduring amplification reactions or in the environment in whichamplification reactions are performed.

The term “amplification conditions” refers to conditions permittingnucleic acid amplification according to the present invention.Amplification conditions may, in some embodiments, be less stringentthan “stringent hybridization conditions” as described herein.Oligonucleotides used in the amplification reactions of the presentinvention hybridize to their intended targets under amplificationconditions, but may or may not hybridize under stringent hybridizationconditions. On the other hand, detection probes of the present inventiontypically hybridize under stringent hybridization conditions. Acceptableconditions to carry out nucleic acid amplifications according to thepresent invention can be easily ascertained by someone having ordinaryskill in the art depending on the particular method of amplificationemployed.

Many well-known methods of nucleic acid amplification requirethermocycling to alternately denature double-stranded nucleic acids andhybridize primers; however, other well-known methods of nucleic acidamplification are isothermal. The polymerase chain reaction (U.S. Pat.Nos. 4,683,195; 4,683,202; 4,800,159; 4,965,188), commonly referred toas PCR, uses multiple cycles of denaturation, annealing of primer pairsto opposite strands, and primer extension to exponentially increase copynumbers of the target sequence. In a variation called RT-PCR, reversetranscriptase (RT) is used to make a complementary DNA (cDNA) from mRNA,and the cDNA is then amplified by PCR to produce multiple copies of DNA.

As noted above, the term “PCR” refers to multiple amplification cyclesthat selectively amplify a target nucleic acid species. Included arequantitative PCR (qPCR), real-time PCR), reverse transcription PCR(RT-PCR) and quantitative reverse transcription PCR (qRT-PCR) is welldescribed in the art. The term “pPCR” refers to quantitative polymerasechain reaction, and the term “qRT-PCR” refers to quantitative reversetranscription polymerase chain reaction. qPCR and qRT-PCR may be used toamplify and simultaneously quantify a targeted cDNA molecule. It enablesboth detection and quantification of a specific sequence in a cDNA pool,such as a selected AARS gene or transcript.

The term “real-time PCR” may use DNA-binding dye to bind to alldouble-stranded (ds) DNA in PCR, causing fluorescence of the dye. Anincrease in DNA product during PCR therefore leads to an increase influorescence intensity and is measured at each cycle, thus allowing DNAconcentrations to be quantified. However, dsDNA dyes such as SYBR Greenwill bind to all dsDNA PCR products. Fluorescence is detected andmeasured in the real-time PCR thermocycler, and its geometric increasecorresponding to exponential increase of the product is used todetermine the threshold cycle (“Ct”) in each reaction.

The term “Ct Score” refers to the threshold cycle number, which is thecycle at which PCR amplification has surpassed a threshold level. Ifthere is a higher quantity of mRNA for a particular gene in a sample, itwill cross the threshold earlier than a lowly expressed gene since thereis more starting RNA to amplify. Therefore, a low Ct score indicateshigh gene expression in a sample and a high Ct score is indicative oflow gene expression.

Certain embodiments may employ the ligase chain reaction (Weiss,Science. 254: 1292, 1991), commonly referred to as LCR, which uses twosets of complementary DNA oligonucleotides that hybridize to adjacentregions of the target nucleic acid. The DNA oligonucleotides arecovalently linked by a DNA ligase in repeated cycles of thermaldenaturation, hybridization and ligation to produce a detectabledouble-stranded ligated oligonucleotide product.

Another method is strand displacement amplification (Walker, G. et al.,1992, Proc. Natl. Acad. Sci. USA 89:392-396; U.S. Pat. Nos. 5,270,184and 5,455,166), commonly referred to as SDA, which uses cycles ofannealing pairs of primer sequences to opposite strands of a targetsequence, primer extension in the presence of a dNTPαS to produce aduplex hemiphosphorothioated primer extension product,endonuclease-mediated nicking of a hemimodified restriction endonucleaserecognition site, and polymerase-mediated primer extension from the 3′end of the nick to displace an existing strand and produce a strand forthe next round of primer annealing, nicking and strand displacement,resulting in geometric amplification of product. Thermophilic SDA (tSDA)uses thermophilic endonucleases and polymerases at higher temperaturesin essentially the same method (European Pat. No. 0 684 315).

Other amplification methods include, for example: nucleic acid sequencebased amplification (U.S. Pat. No. 5,130,238), commonly referred to asNASBA; one that uses an RNA replicase to amplify the probe moleculeitself (Lizardi, P. et al., 1988, BioTechnol. 6: 1197-1202), commonlyreferred to as Qβ replicase; a transcription based amplification method(Kwoh, D. et al., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177);self-sustained sequence replication (Guatelli, J. et al., 1990, Proc.Natl. Acad. Sci. USA 87: 1874-1878); and, transcription mediatedamplification (U.S. Pat. Nos. 5,480,784 and 5,399,491), commonlyreferred to as TMA. For further discussion of known amplificationmethods see Persing, David H., 1993, “In Vitro Nucleic AcidAmplification Techniques” in Diagnostic Medical Microbiology: Principlesand Applications (Persing et al., Eds.), pp. 51-87 (American Society forMicrobiology, Washington, DC).

Illustrative transcription-based amplification systems of the presentinvention include TMA, which employs an RNA polymerase to producemultiple RNA transcripts of a target region (U.S. Pat. Nos. 5,480,784and 5,399,491). TMA uses a “promoter-primer” that hybridizes to a targetnucleic acid in the presence of a reverse transcriptase and an RNApolymerase to form a double-stranded promoter from which the RNApolymerase produces RNA transcripts. These transcripts can becometemplates for further rounds of TMA in the presence of a second primercapable of hybridizing to the RNA transcripts. Unlike PCR, LCR or othermethods that require heat denaturation, TMA is an isothermal method thatuses an RNase H activity to digest the RNA strand of an RNA:DNA hybrid,thereby making the DNA strand available for hybridization with a primeror promoter-primer. Generally, the RNase H activity associated with thereverse transcriptase provided for amplification is used.

In an illustrative TMA method, one amplification primer is anoligonucleotide promoter-primer that comprises a promoter sequence whichbecomes functional when double-stranded, located 5′ of a target-bindingsequence, which is capable of hybridizing to a binding site of a targetRNA at a location 3′ to the sequence to be amplified. A promoter-primermay be referred to as a “T7-primer” when it is specific for T7 RNApolymerase recognition. Under certain circumstances, the 3′ end of apromoter-primer, or a subpopulation of such promoter-primers, may bemodified to block or reduce primer extension. From an unmodifiedpromoter-primer, reverse transcriptase creates a cDNA copy of the targetRNA, while RNase H activity degrades the target RNA. A secondamplification primer then binds to the cDNA. This primer may be referredto as a “non-T7 primer” to distinguish it from a “T7-primer.” From thissecond amplification primer, reverse transcriptase creates another DNAstrand, resulting in a double-stranded DNA with a functional promoter atone end. When double-stranded, the promoter sequence is capable ofbinding an RNA polymerase to begin transcription of the target sequenceto which the promoter-primer is hybridized. An RNA polymerase uses thispromoter sequence to produce multiple RNA transcripts (i.e., amplicons),generally about 100 to 1,000 copies. Each newly-synthesized amplicon cananneal with the second amplification primer. Reverse transcriptase canthen create a DNA copy, while the RNase H activity degrades the RNA ofthis RNA:DNA duplex. The promoter-primer can then bind to the newlysynthesized DNA, allowing the reverse transcriptase to create adouble-stranded DNA, from which the RNA polymerase produces multipleamplicons. Thus, a billion-fold isothermic amplification can be achievedusing two amplification primers.

In certain embodiments, other techniques may be used to evaluate RNAtranscripts of the transcripts from a particular cDNA library, includingmicroarray analysis (Han, M., et al., Nat Biotechnol, 19: 631-635, 2001;Bao, P., et al., Anal Chem, 74: 1792-1797, 2002; Schena et al., Proc.Natl. Acad. Sci. USA 93:10614-19, 1996; and Heller et al., Proc. Natl.Acad. Sci. USA 94:2150-55, 1997) and SAGE (serial analysis of geneexpression). Like MPSS, SAGE is digital and can generate a large numberof signature sequences. (see e.g., Velculescu, V. E., et al., TrendsGenet, 16: 423-425., 2000; Tuteja R. and Tuteja N. Bioessays. 2004August; 26(8):916-22), although orders of magnitude fewer than that areavailable from techniques such as MPSS.

In certain embodiments, the term “microarray” includes a “nucleic acidmicroarray” having a substrate-bound plurality of nucleic acids,hybridization to each of the plurality of bound nucleic acids beingseparately detectable. The substrate can be solid or porous, planar ornon-planar, unitary or distributed. Nucleic acid microarrays include allthe devices so called in Schena (ed.), DNA Microarrays: A PracticalApproach (Practical Approach Series), Oxford University Press (1999);Nature Genet. 21(1) (suppl.): 1-60 (1999); Schena (ed.), MicroarrayBiochip: Tools and Technology, Eaton Publishing Company/BioTechniquesBooks Division (2000). Nucleic acid microarrays may include asubstrate-bound plurality of nucleic acids in which the plurality ofnucleic acids are disposed on a plurality of beads, rather than on aunitary planar substrate, as described, for example, in Brenner et al.,Proc. Natl. Acad. Sci. USA 97(4): 1665-1670 (2000). Examples of nucleicacid microarrays may be found in U.S. Pat. Nos. 6,391,623, 6,383,754,6,383,749, 6,380,377, 6,379,897, 6,376,191, 6,372,431, 6,351,7126,344,316, 6,316,193, 6,312,906, 6,309,828, 6,309,824, 6,306,643,6,300,063, 6,287,850, 6,284,497, 6,284,465, 6,280,954, 6,262,216,6,251,601, 6,245,518, 6,263,287, 6,251,601, 6,238,866, 6,228,575,6,214,587, 6,203,989, 6,171,797, 6,103,474, 6,083,726, 6,054,274,6,040,138, 6,083,726, 6,004,755, 6,001,309, 5,958,342, 5,952,180,5,936,731, 5,843,655, 5,814,454, 5,837,196, 5,436,327, 5,412,087, and5,405,783, the disclosures of which are incorporated by reference.

Additional examples include nucleic acid arrays that are commerciallyavailable from Affymetrix (Santa Clara, Calif.) under the brand nameGENECHIP™. Further exemplary methods of manufacturing and using arraysare provided in, for example, U.S. Pat. Nos. 7,028,629; 7,011,949;7,011,945; 6,936,419; 6,927,032; 6,924,103; 6,921,642; and 6,818,394.

The present invention as related to arrays and microarrays alsocontemplates many uses for polymers attached to solid substrates. Theseuses include gene expression monitoring, profiling, library screening,genotyping and diagnostics. Gene expression monitoring and profilingmethods and methods useful for gene expression monitoring and profilingare shown in U.S. Pat. Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860,6,040,138, 6,177,248 and 6,309,822. Genotyping and uses therefore areshown in U.S. Ser. Nos. 10/442,021, 10/013,598 (U.S. Application No.2003/0036069), and U.S. Pat. Nos. 5,925,525, 6,268,141, 5,856,092,6,267,152, 6,300,063, 6,525,185, 6,632,611, 5,858,659, 6,284,460,6,361,947, 6,368,799, 6,673,579 and 6,333,179. Other methods of nucleicacid amplification, labeling and analysis that may be used incombination with the methods disclosed herein are embodied in U.S. Pat.Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061, and 6,197,506.

As will be apparent to persons skilled in the art, certain embodimentsmay employ oligonucleotides, such as primers or probes, foramplification or detection, as described herein. Oligonucleotides of adefined sequence and chemical structure may be produced by techniquesknown to those of ordinary skill in the art, such as by chemical orbiochemical synthesis, and by in vitro or in vivo expression fromrecombinant nucleic acid molecules, e.g., bacterial or viral vectors. Incertain embodiments, an oligonucleotide does not consist solely ofwild-type chromosomal DNA or the in vivo transcription products thereof.

Oligonucleotides or primers may be modified in any way, as long as agiven modification is compatible with the desired function of a givenoligonucleotide. One of ordinary skill in the art can easily determinewhether a given modification is suitable or desired for any givenoligonucleotide of the present invention. Relevant AARS oligonucleotidesare described in greater detail elsewhere herein.

While the design and sequence of oligonucleotides depends on theirfunction as described herein, several variables are generally taken intoaccount. Among the most relevant are: length, melting temperature (Tm),specificity, complementarity with other oligonucleotides in the system,G/C content, polypyrimidine (T, C) or polypurine (A, G) stretches, andthe 3′-end sequence. Controlling for these and other variables is astandard and well known aspect of oligonucleotide design, and variouscomputer programs are readily available to screen large numbers ofpotential oligonucleotides for optimal ones.

Certain embodiments therefore include methods for detecting a targetAARS polynucleotide in a sample, the polynucleotide comprising thesequence of a reference AARS polynucleotide, as described herein,comprising a) hybridizing the sample with a probe comprising a sequencecomplementary to the target polynucleotide in the sample, and whichprobe specifically hybridizes to said target polynucleotide, underconditions whereby a hybridization complex is formed between said probeand said target polynucleotide or fragments thereof, and b) detectingthe presence or absence of said hybridization complex, and optionally,if present, the amount thereof. Also included are methods for detectinga target AARS polynucleotide in a sample, the polynucleotide comprisingthe sequence of a reference AARS polynucleotide, as described herein,comprising a) amplifying the target polynucleotide or fragment thereof,and b) detecting the presence or absence of said amplified targetpolynucleotide or fragment thereof, and, optionally, if present, theamount thereof. Specific embodiments relate to the detection of AARSsplice variants, such as by detecting a unique splice junction of thesplice variant, whether by hybridization, amplification, or otherdetection method.

Embodiments of the present invention include a variety of AARSpolypeptide-based detection techniques, including antibody-baseddetection techniques. Included in these embodiments are the use of AARSpolypeptides to generate antibodies or other binders, which may then beused in diagnostic methods and compositions to detect or quantitateselected AARS polypeptides in a cell or other biological sample,typically from a subject.

Certain embodiments may employ standard methodologies and detectors suchas western blotting and immunoprecipitation, enzyme-linked immunosorbentassays (ELISA), flow cytometry, and immunofluorescence assays (IFA),which utilize an imaging device. These well-known methods typicallyutilize one or more monoclonal or polyclonal antibodies as describedherein that specifically bind to a selected AARS polypeptide of theinvention, or a unique region of that AARS polypeptide, and generally donot bind significantly to other AARS polypeptides, such as a full-lengthAARS polypeptide. In certain embodiments, the unique region of the AARSpolypeptide may represent a unique three-dimensional structure that ispossessed by a newly identified protein fragment of an AARS.

Certain embodiments may employ “arrays,” such as “microarrays.” Incertain embodiments, a “microarray” may also refer to a “peptidemicroarray” or “protein microarray” having a substrate-bound collectionor plurality of polypeptides, the binding to each of the plurality ofbound polypeptides being separately detectable. Alternatively, thepeptide microarray may have a plurality of binders, including but notlimited to monoclonal antibodies, polyclonal antibodies, phage displaybinders, yeast 2 hybrid binders, and aptamers, which can specificallydetect the binding of the AARS polypeptides described herein. The arraymay be based on autoantibody detection of these AARS polypeptides, asdescribed, for example, in Robinson et al., Nature Medicine 8(3):295-301(2002). Examples of peptide arrays may be found in WO 02/31463, WO02/25288, WO 01/94946, WO 01/88162, WO 01/68671, WO 01/57259, WO00/61806, WO 00/54046, WO 00/47774, WO 99/40434, WO 99/39210, and WO97/42507 and U.S. Pat. Nos. 6,268,210, 5,766,960, and 5,143,854, each ofwhich are incorporated by reference.

Certain embodiments may employ MS or other molecular weight-basedmethods for diagnostically detecting AARS polypeptide sequences. Massspectrometry (MS) refers generally to an analytical technique fordetermining the elemental composition of a sample or molecule. MS mayalso be used for determining the chemical structures of molecules, suchas peptides and other chemical compounds.

Generally, the MS principle consists of ionizing chemical compounds togenerate charged molecules or molecule fragments, and then measuringtheir mass-to-charge ratios. In an illustrative MS procedure: a sampleis loaded onto the MS instrument, and undergoes vaporization, thecomponents of the sample are ionized by one of a variety of methods(e.g., by impacting them with an electron beam), which results in theformation of positively charged particles, the positive ions are thenaccelerated by a magnetic field, computations are performed on themass-to-charge ratio (m/z) of the particles based on the details ofmotion of the ions as they transit through electromagnetic fields, and,detection of the ions, which in step prior were sorted according to m/z.

An illustrative MS instruments has three modules: an ion source, whichconverts gas phase sample molecules into ions (or, in the case ofelectrospray ionization, move ions that exist in solution into the gasphase); a mass analyzer, which sorts the ions by their masses byapplying electromagnetic fields; and a detector, which measures thevalue of an indicator quantity and thus provides data for calculatingthe abundances of each ion present.

The MS technique has both qualitative and quantitative uses, includingidentifying unknown compounds, determining the isotopic composition ofelements in a molecule, and determining the structure of a compound byobserving its fragmentation. Other uses include quantifying the amountof a compound in a sample or studying the fundamentals of gas phase ionchemistry (the chemistry of ions and neutrals in a vacuum). Included aregas chromatography-mass spectrometry (GC/MS or GC-MS), liquidchromatography mass spectrometry (LC/MS or LC-MS), and ion mobilityspectrometry/mass spectrometry (IMS/MS or IMMS) Accordingly, MStechniques may be used according to any of the methods provided hereinto measure the presence or levels of an AARS polypeptide of theinvention in a biological sample, and to compare those levels to acontrol sample or a pre-determined value.

Certain embodiments may employ cell-sorting or cell visualization orimaging devices/techniques to detect or quantitate the presence orlevels of AARS polynucleotides or polypeptides. Examples include flowcytometry or FACS, immunofluorescence analysis (IFA), and in situhybridization techniques, such as fluorescent in situ hybridization(FISH).

Certain embodiments may employ conventional biology methods, softwareand systems for diagnostic purposes. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, forexample Setubal and Meidanis et al., Introduction to ComputationalBiology Methods (PWS Publishing Company, Boston, 1997); Salzberg,Searles, Kasif, (Ed.), Computational Methods in Molecular Biology,(Elsevier, Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2nd ed., 2001). See U.S. Pat.No. 6,420,108.

Certain embodiments may employ various computer program products andsoftware for a variety of purposes, such as probe design, management ofdata, analysis, and instrument operation. See, U.S. Pat. Nos. 5,593,839,5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555, 6,185,561,6,188,783, 6,223,127, 6,229,911 and 6,308,170.

The whole genome sampling assay (WGSA) is described, for example inKennedy et al., Nat. Biotech. 21, 1233-1237 (2003), Matsuzaki et al.,Gen. Res. 14: 414-425, (2004), and Matsuzaki, et al., Nature Methods1:109-111 (2004). Algorithms for use with mapping assays are described,for example, in Liu et al., Bioinformatics. 19: 2397-2403 (2003) and Diet al. Bioinformatics. 21:1958 (2005). Additional methods related toWGSA and arrays useful for WGSA and applications of WGSA are disclosed,for example, in U.S. Patent Application No. 60/676,058 filed Apr. 29,2005, 60/616,273 filed Oct. 5, 2004, Ser. Nos. 10/912,445, 11/044,831,10/442,021, 10/650,332 and 10/463,991. Genome wide association studiesusing mapping assays are described in, for example, Hu et al., CancerRes.; 65(7):2542-6 (2005), Mitra et al., Cancer Res., 64(21):8116-25(2004), Butcher et al., Hum Mol Genet., 14(10):1315-25 (2005), and Kleinet al., Science. 308(5720):385-9 (2005).

Additionally, certain embodiments may include methods for providinggenetic information over networks such as the Internet as shown, forexample, in U.S. application Ser. Nos. 10/197,621, 10/063,559 (UnitedStates Publication Number 2002/0183936), Ser. Nos. 10/065,856,10/065,868, 10/328,818, 10/328,872, 10/423,403, and 60/482,389.

X. ANTISENSE AND RNAI AGENTS

Embodiments of the present invention also include antisenseoligonucleotides and RNAi agents that target the AARS polynucleotidesequences, and methods of use thereof to reduce expression of a selectedAARS transcript and/or protein fragment. Certain embodiments relate totargeting one or more splice junctions (often unique) that generate asplice variant, AARS protein fragment of instant invention. Alsoincluded are methods of antisense or RNAi inhibition that target certainsplice forms, either to encourage or discourage splicing of a selectedprotein fragment. In certain preferred embodiments, the splice junctionsthat generate the AARS protein fragments are over-expressed with respectto particular tissues, and are unique to that splice variant. In theseand related embodiments, such splice variants are not the only source ofcytosolic AARS activity in the targeted cell type. For instance, certainsplice variants to be targeted may represent about 10% to 50% of thetotal copy number of the AARS RNA splice variants in a given cell ortissue, and preferably about 1-10% of the total copy number of the AARSRNA splice variants in a given cell or tissue. Splice variants that areabout <1% of the total copy number of the AARS RNA splice variants in agiven cell or tissue may also be targeted.

In certain embodiments, the antisense or RNAi agent does not target thefull-length protein, because such full-length proteins are responsiblefor a key step in protein synthesis, and thereby avoids lethality thatoften results from wild-type AARS knockouts. Certain of the methodsdescribed herein can therefore by used to avoid undesired effects suchas toxicities in both chronic and acute treatments, and to selectivelymodulate the non-canonical activities of the AARS protein fragment.However, certain embodiments may generically target AARS sequences,including full-length AARS sequences, such as to kill or substantiallyderange the cell physiology of a target cell or tissue.

In certain embodiments, the AARS splice variant to be targeted possessesa non-canonical biological activity. In some embodiments, the AARSsplice variant has reduced or undetectable canonical AARS activity, andthe antisense or RNAi-related method more specifically modulates itsnon-canonical activity. In certain embodiments, the antisense orRNAi-related agents can be combined with a targeted or local deliveryapproach to lessen systemic undesired effects to non-targeted cells ortissues. Among others described herein, exemplary cells or tissues thatcould be targeted this way include cancer cells, and cells to tissuesthat lend themselves to localized targeting, such as tumors or epitheliavia topical application.

A. Antisense Agents

The terms “antisense oligomer” or “antisense compound” or “antisenseoligonucleotide” are used interchangeably and refer to a sequence ofcyclic subunits, each bearing a base-pairing moiety, linked byintersubunit linkages that allow the base-pairing moieties to hybridizeto a target sequence in a nucleic acid (typically an RNA) byWatson-Crick base pairing, to form a nucleic acid:oligomer heteroduplexwithin the target sequence, and typically thereby prevent translation ofthat RNA. Also included are methods of use thereof to modulateexpression of a selected AARS transcript, such as a splice variant orproteolytic fragment, and/or its corresponding polypeptide.

Antisense oligonucleotides may contain between about 8 and 40 subunits,typically about 8-25 subunits, and preferably about 12 to 25 subunits.In certain embodiments, oligonucleotides may have exact sequencecomplementarity to the target sequence or near complementarity, asdefined below. In certain embodiments, the degree of complementaritybetween the target and antisense targeting sequence is sufficient toform a stable duplex. The region of complementarity of the antisenseoligomers with the target RNA sequence may be as short as 8-11 bases,but is preferably 12-15 bases or more, e.g., 12-20 bases, or 12-25bases, including all integers in between these ranges. An antisenseoligomer of about 14-15 bases is generally long enough to have a uniquecomplementary sequence in targeting the selected AARS gene. In certainembodiments, a minimum length of complementary bases may be required toachieve the requisite binding Tm, as discussed herein.

In certain embodiments, antisense oligomers as long as 40 bases may besuitable, where at least a minimum number of bases, e.g., 10-12 bases,are complementary to the target sequence. In general, however,facilitated or active uptake in cells is optimized at oligomer lengthsless than about 30. For certain oligomers, described further below, anoptimum balance of binding stability and uptake generally occurs atlengths of 18-25 bases. Included are antisense oligomers (e.g., PNAs,LNAs, 2′-OMe, MOE) that consist of about 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, or 40 bases, in which at least about 6, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous ornon-contiguous bases are complementary to their AARS target sequence, orvariants thereof.

In certain embodiments, antisense oligomers may be 100% complementary toan AARS nucleic acid target sequence, or it may include mismatches,e.g., to accommodate variants, as long as a heteroduplex formed betweenthe oligomer and AARS nucleic acid target sequence is sufficientlystable to withstand the action of cellular nucleases and other modes ofdegradation which may occur in vivo. The term “target sequence” refersto a portion of the target RNA against which the oligonucleotide isdirected, that is, the sequence to which the oligonucleotide willhybridize by Watson-Crick base pairing of a complementary sequence. Incertain embodiments, the target sequence may be a contiguous region ofan AARS mRNA (e.g., a unique splice junction of an AARS mRNA), or may becomposed of non-contiguous regions of the mRNA.

Oligomer backbones which are less susceptible to cleavage by nucleasesare discussed below. Mismatches, if present, are less destabilizingtoward the end regions of the hybrid duplex than in the middle. Thenumber of mismatches allowed will depend on the length of the oligomer,the percentage of G:C base pairs in the duplex, and the position of themismatch(es) in the duplex, according to well understood principles ofduplex stability. Although such an antisense oligomer is not necessarily100% complementary to the AARS nucleic acid target sequence, it iseffective to stably and specifically bind to the target sequence, suchthat a biological activity of the nucleic acid target, e.g., expressionof AARS protein(s), is modulated.

The stability of the duplex formed between an oligomer and a targetsequence is a function of the binding Tm and the susceptibility of theduplex to cellular enzymatic cleavage. The Tm of an antisenseoligonucleotide with respect to complementary-sequence RNA may bemeasured by conventional methods, such as those described by Hames etal., Nucleic Acid Hybridization, IRL Press, 1985, pp. 107-108 or asdescribed in Miyada C. G. and Wallace R. B., 1987, Oligonucleotidehybridization techniques, Methods Enzymol. Vol. 154 pp. 94-107. Incertain embodiments, antisense oligomer may have a binding Tm, withrespect to a complementary-sequence RNA, of greater than bodytemperature and preferably greater than 50° C. Tm's in the range 60-80°C. or greater are preferred. According to well known principles, the Tmof an oligomer compound, with respect to a complementary-based RNAhybrid, can be increased by increasing the ratio of C:G paired bases inthe duplex, and/or by increasing the length (in base pairs) of theheteroduplex. At the same time, for purposes of optimizing cellularuptake, it may be advantageous to limit the size of the antisenseoligomer. For this reason, compounds that show high Tm (50° C. orgreater) at a length of 25 bases or less are generally preferred overthose requiring greater than 25 bases for high Tm values.

Antisense oligomers can be designed to block or inhibit translation ofmRNA or to inhibit natural pre-mRNA splice processing, or inducedegradation of targeted mRNAs, and may be said to be “directed to” or“targeted against” a target sequence with which it hybridizes. Incertain embodiments, the target sequence may include any coding ornon-coding sequence of an AARS mRNA transcript, and may thus by withinan exon or within an intron. In certain embodiments, the target sequenceis relatively unique or exceptional among AARSs (e.g., a full-lengthAARS) and is selective for reducing expression of a selected AARSprotein fragment, such as a proteolytic fragment or splice variant. Incertain embodiments, the target site includes a 3′ or 5′ splice site ofa pre-processed mRNA, or a branch point. The target sequence for asplice site may include an mRNA sequence having its 5′ end 1 to about 25to about 50 base pairs downstream of a splice acceptor junction orupstream of a splice donor junction in a preprocessed mRNA. In certainembodiments, a target sequence may include a splice junction of analternatively splice AARS mRNA, such as a splice junction that does notoccur in the full-length AARS, or is unique or exceptional to thattranscript, in that it either does not occur or only seldom occurs inother AARS splice variants. An oligomer is more generally said to be“targeted against” a biologically relevant target, such as referenceAARS polynucleotide, when it is targeted against the nucleic acid of thetarget in the manner described herein.

An oligonucleotide is typically complementary to a target sequence, suchas a target DNA or RNA. The terms “complementary” and “complementarity”refer to polynucleotides (i.e., a sequence of nucleotides) related bythe base-pairing rules. For example, the sequence “A-G-T,” iscomplementary to the sequence “T-C-A.” Complementarity may be “partial,”in which only some of the nucleic acids' bases are matched according tothe base pairing rules. Or, there may be “complete” or “total”complementarity (100%) between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. While perfect complementarity is often desired, someembodiments can include one or more but preferably 20, 19, 18, 17, 16,15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatches withrespect to the target sequence. Variations at any location within theoligomer are included. In certain embodiments, variations in sequencenear the termini of an oligomer are generally preferable to variationsin the interior, and if present are typically within about 10, 9, 8, 7,6, 5, 4, 3, 2, or 1 nucleotides of the 5′ and/or 3′ terminus.

The term “targeting sequence” or in certain embodiments “antisensetargeting sequence” refers to the sequence in an oligonucleotide that iscomplementary (meaning, in addition, substantially complementary) to thetarget sequence in the DNA or RNA target molecule. The entire sequence,or only a portion, of the antisense compound may be complementary to thetarget sequence. For example, in an oligonucleotide having 20-30 bases,about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, or 29 may be targeting sequences that arecomplementary to the target region. Typically, the targeting sequence isformed of contiguous bases, but may alternatively be formed ofnon-contiguous sequences that when placed together, e.g., from oppositeends of the oligonucleotide, constitute sequence that spans the targetsequence.

Target and targeting sequences are described as “complementary” to oneanother when hybridization occurs in an antiparallel configuration. Atargeting sequence may have “near” or “substantial” complementarity tothe target sequence and still function for the purpose of the presentinvention, that is, it may still be functionally “complementary.” Incertain embodiments, an oligonucleotide may have at most one mismatchwith the target sequence out of 10 nucleotides, and preferably at mostone mismatch out of 20. Alternatively, an oligonucleotide may have atleast about 80%, 85%, 90% sequence homology, and preferably at least 95%sequence homology, with an AARS reference polynucleotide sequencedescribed herein, or its complement.

An oligonucleotide “specifically hybridizes” to a target polynucleotideif the oligomer hybridizes to a target (e.g., an AARS referencepolynucleotide or its complement) under physiological conditions, with aTm substantially greater than 45° C., preferably at least 50° C., andtypically 60° C.-80° C. or higher. Such hybridization preferablycorresponds to stringent hybridization conditions. At a given ionicstrength and pH, the Tm is the temperature at which 50% of a targetsequence hybridizes to a complementary polynucleotide. Again, suchhybridization may occur with “near” or “substantial” complementarity ofthe antisense oligomer to the target sequence, as well as with exactcomplementarity.

The terms specifically binds or specifically hybridizes refer generallyto an oligonucleotide probe or polynucleotide sequence that not onlybinds to its intended target gene sequence in a sample under selectedhybridization conditions, but does not bind significantly to othertarget sequences in the sample, and thereby discriminates between itsintended target and all other targets in the target pool. A probe thatspecifically hybridizes to its intended target sequence may also detectconcentration differences under the selected hybridization conditions,as described herein.

A “nuclease-resistant” oligomeric molecule (oligomer) refers to onewhose backbone is substantially resistant to nuclease cleavage, innon-hybridized or hybridized form; by common extracellular andintracellular nucleases in the body; that is, the oligomer shows littleor no nuclease cleavage under normal nuclease conditions in the body towhich the oligomer is exposed.

A “heteroduplex” refers to a duplex between an oligonucleotide and thecomplementary portion of a target polynucleotide, such as a target DNAor RNA. A “nuclease-resistant heteroduplex” refers to a heteroduplexformed by the binding of an oligomer to its complementary target, suchthat the heteroduplex is substantially resistant to in vivo degradationby intracellular and extracellular nucleases, such as RNaseH, which arecapable of cutting double-stranded RNA/RNA or RNA/DNA complexes.

A “subunit” of an oligonucleotide refers to one nucleotide (ornucleotide analog) unit. The term may refer to the nucleotide unit withor without the attached intersubunit linkage, although, when referringto a “charged subunit”, the charge typically resides within theintersubunit linkage (e.g., a phosphate or phosphorothioate linkage or acationic linkage).

The cyclic subunits of an oligonucleotide may be based on ribose oranother pentose sugar or, in certain embodiments, alternate or modifiedgroups. Examples of modified oligonucleotide backbones include, withoutlimitation, phosphorothioates, chiral phosphorothioates,phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,methyl and other alkyl phosphonates including 3′-alkylene phosphonatesand chiral phosphonates, phosphinates, phosphoramidates including3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′linkages, 2′-5′ linked analogs of these, and those having invertedpolarity wherein the adjacent pairs of nucleoside units are linked 3′-5′to 5′-3′ or 2′-5′ to 5′-2′. Also contemplated are peptide nucleic acids(PNAs), locked nucleic acids (LNAs), 2′-O-Methyl oligonucleotides(2′-OMe), 2′-methoxyethoxy oligonucleotides (MOE), among otheroligonucleotides known in the art.

The purine or pyrimidine base pairing moiety is typically adenine,cytosine, guanine, uracil, thymine or inosine. Also included are basessuch as pyridin-4-one, pyridin-2-one, phenyl, pseudouracil,2,4,6-trime115thoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl,aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines(e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or6-azapyrimidines or 6-alkylpyrimidines (e.g. 6-methyluridine), propyne,quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine,4-acetyltidine, 5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, β-D-galactosylqueosine,1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonyhnethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine,β-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra). By “modified bases” in this aspect ismeant nucleotide bases other than adenine (A), guanine (G), cytosine(C), thymine (T), and uracil (U), as illustrated above; such bases canbe used at any position in the antisense molecule. Persons skilled inthe art will appreciate that depending on the uses of the oligomers, Tsand Us are interchangeable. For instance, with other antisensechemistries such as 2′-O-methyl antisense oligonucleotides that are moreRNA-like, the T bases may be shown as U.

As noted above, certain oligonucleotides provided herein include peptidenucleic acids (PNAs). Peptide nucleic acids (PNAs) are analogs of DNA inwhich the backbone is structurally homomorphous with a deoxyribosebackbone, consisting of N-(2-aminoethyl) glycine units to whichpyrimidine or purine bases are attached. PNAs containing naturalpyrimidine and purine bases hybridize to complementary oligonucleotidesobeying Watson-Crick base-pairing rules, and mimic DNA in terms of basepair recognition (Egholm, Buchardt et al. 1993). The backbone of PNAs isformed by peptide bonds rather than phosphodiester bonds, making themwell-suited for antisense applications (see structure below). Thebackbone is uncharged, resulting in PNA/DNA or PNA/RNA duplexes thatexhibit greater than normal thermal stability. PNAs are not recognizedby nucleases or proteases.

PNAs may be produced synthetically using any technique known in the art.PNA is a DNA analog in which a polyamide backbone replaces thetraditional phosphate ribose ring of DNA. Despite a radical structuralchange to the natural structure, PNA is capable of sequence-specificbinding in a helix form to DNA or RNA. Characteristics of PNA include ahigh binding affinity to complementary DNA or RNA, a destabilizingeffect caused by single-base mismatch, resistance to nucleases andproteases, hybridization with DNA or RNA independent of saltconcentration and triplex formation with homopurine DNA. Panagene™ hasdeveloped its proprietary Bts PNA monomers (Bts;benzothiazole-2-sulfonyl group) and proprietary oligomerisation process.The PNA oligomerisation using Bts PNA monomers is composed of repetitivecycles of deprotection, coupling and capping. Panagene's patents to thistechnology include U.S. Pat. No. 6,969,766, U.S. Pat. No. 7,211,668,U.S. Pat. No. 7,022,851, U.S. Pat. No. 7,125,994, U.S. Pat. No.7,145,006 and U.S. Pat. No. 7,179,896. Representative United Statespatents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each ofwhich is herein incorporated by reference. Further teaching of PNAcompounds can be found in Nielsen et al., Science, 1991, 254, 1497.

Also included are “locked nucleic acid” subunits (LNAs). The structuresof LNAs are known in the art: for example, Wengel, et al., ChemicalCommunications (1998) 455; Tetrahedron (1998) 54, 3607, and Accounts ofChem. Research (1999) 32, 301); Obika, et al., Tetrahedron Letters(1997) 38, 8735; (1998) 39, 5401, and Bioorganic Medicinal Chemistry(2008)16, 9230.

Oligonucleotides may incorporate one or more LNAs; in some cases, thecompounds may be entirely composed of LNAs. Methods for the synthesis ofindividual LNA nucleoside subunits and their incorporation intooligonucleotides are known in the art: U.S. Pat. Nos. 7,572,582;7,569,575; 7,084,125; 7,060,809; 7,053,207; 7,034,133; 6,794,499; and6,670,461. Typical intersubunit linkers include phosphodiester andphosphorothioate moieties; alternatively, non-phosphorous containinglinkers may be employed. A preferred embodiment is an LNA containingcompound where each LNA subunit is separated by a DNA subunit (i.e., adeoxyribose nucleotide). Further preferred compounds are composed ofalternating LNA and DNA subunits where the intersubunit linker isphosphorothioate.

Certain oligonucleotides may comprise morpholino-based subunits bearingbase-pairing moieties, joined by uncharged or substantially unchargedlinkages. The terms “morpholino oligomer” or “PMO” (phosphoramidate- orphosphorodiamidate morpholino oligomer) refer to an oligonucleotideanalog composed of morpholino subunit structures, where (i) thestructures are linked together by phosphorus-containing linkages, one tothree atoms long, preferably two atoms long, and preferably uncharged orcationic, joining the morpholino nitrogen of one subunit to a 5′exocyclic carbon of an adjacent subunit, and (ii) each morpholino ringbears a purine or pyrimidine or an equivalent base-pairing moietyeffective to bind, by base specific hydrogen bonding, to a base in apolynucleotide.

Variations can be made to this linkage as long as they do not interferewith binding or activity. For example, the oxygen attached to phosphorusmay be substituted with sulfur (thiophosphorodiamidate). The 5′ oxygenmay be substituted with amino or lower alkyl substituted amino. Thependant nitrogen attached to phosphorus may be unsubstituted,monosubstituted, or disubstituted with (optionally substituted) loweralkyl. The purine or pyrimidine base pairing moiety is typicallyadenine, cytosine, guanine, uracil, thymine or inosine. The synthesis,structures, and binding characteristics of morpholino oligomers aredetailed in U.S. Pat. Nos. 5,698,685, 5,217,866, 5,142,047, 5,034,506,5,166,315, 5,521,063, and 5,506,337, and PCT Appn. Nos. PCT/US07/11435(cationic linkages) and U.S. Ser. No. 08/012,804 (improved synthesis),all of which are incorporated herein by reference.

The morpholino subunits may also be linked by non-phosphorus-basedintersubunit linkages, as described further below, where at least onelinkage is modified with a pendant cationic group as described above.Other oligonucleotide analog linkages which are uncharged in theirunmodified state but which could also bear a pendant amine substituentcould be used. For example, a 5′nitrogen atom on a morpholino ring couldbe employed in a sulfamide linkage or a urea linkage (where phosphorusis replaced with carbon or sulfur, respectively) and modified in amanner analogous to the 5′-nitrogen atom in structure (b3) above

Certain embodiments include substantially uncharged morpholinooligomers, such as a substantially uncharged phosphorodiamidate-linkedmorpholino oligomer. A substantially uncharged, phosphorus containingbackbone in an oligonucleotide analog is one in which a majority of thesubunit linkages, e.g., between 50-100%, typically at least 60% to 100%or 75% or 80% of its linkages, are uncharged at physiological pH, andcontain a single phosphorous atom. Examples of morpholinooligonucleotides having phosphorus-containing backbone linkages includephosphoroamidate and phosphorodiamidate-linked morpholinooligonucleotides. Certain embodiments may contain positively chargedgroups at preferably about 10%-50% of their backbone linkages.

Properties of the morpholino-based subunits include, for example, theability to be linked in a oligomeric form by stable, uncharged orpositively charged backbone linkages, the ability to support anucleotide base (e.g., adenine, cytosine, guanine, thymidine, uracil andhypoxanthine) such that the polymer formed can hybridize with acomplementary-base target nucleic acid, including target RNA, Tm valuesabove about 45° C. in relatively short oligonucleotides (e.g., 10-15bases), the ability of the oligonucleotide to be actively or passivelytransported into mammalian cells, and the ability of the antisenseoligonucleotide:RNA heteroduplex to resist RNase and RNaseH degradation,respectively.

In certain embodiments, a substantially uncharged oligonucleotide may bemodified to include charged linkages, e.g., up to about 1 per every 2-5uncharged linkages, such as about 4-5 per every 10 uncharged linkages.In certain embodiments, optimal improvement in antisense activity may beseen when about 25% of the backbone linkages are cationic. In certainembodiments, enhancement may be seen with a small number e.g., 10-20%cationic linkages, or where the number of cationic linkages are in therange 50-80%, such as about 60%. In certain embodiments the cationicbackbone charges may be further enhanced by distributing the bulk of thecharges close of the “center-region” backbone linkages of the antisenseoligonucleotide, e.g., in a 20-mer oligonucleotide with 8 cationicbackbone linkages, having at least 70% of these charged linkageslocalized in the 10 centermost linkages.

Oligonucleotides that target one or more portions of an AARSpolynucleotide reference sequence or its complement may be used in anyof the therapeutic, diagnostic, or drug screening methods describedherein and apparent to persons skilled in the art.

B. RNA Interference Agents

Certain embodiments relate to RNA interference (RNAi) agents that targetone or more mRNA transcripts of an aminoacyl-tRNA synthetase (AARS)reference polynucleotide, including fragments and splice variantsthereof. Also included are methods of use thereof to modulate the levelsof a selected AARS transcript, such as an AARS splice variant orendogenous proteolytic fragment.

The term “double-stranded” means two separate nucleic acid strandscomprising a region in which at least a portion of the strands aresufficiently complementary to hydrogen bond and form a duplex structure.The term “duplex” or “duplex structure” refers to the region of a doublestranded molecule wherein the two separate strands are substantiallycomplementary, and thus hybridize to each other. “dsRNA” refers to aribonucleic acid molecule having a duplex structure comprising twocomplementary and anti-parallel nucleic acid strands (i.e., the senseand antisense strands). Not all nucleotides of a dsRNA must exhibitWatson-Crick base pairs; the two RNA strands may be substantiallycomplementary. The RNA strands may have the same or a different numberof nucleotides.

In certain embodiments, a dsRNA is or includes a region which is atleast partially complementary to the target RNA. In certain embodiments,the dsRNA is fully complementary to the target RNA. It is not necessarythat there be perfect complementarity between the dsRNA and the target,but the correspondence must be sufficient to enable the dsRNA, or acleavage product thereof, to direct sequence specific silencing, such asby RNAi cleavage of the target RNA. Complementarity, or degree ofhomology with the target strand, is typically most critical in theantisense strand. While perfect complementarity, particularly in theantisense strand, is often desired some embodiments can include one ormore but preferably 6, 5, 4, 3, 2, or fewer mismatches with respect tothe target RNA. The mismatches are most tolerated in the terminalregions, and if present are preferably in a terminal region or regions,e.g., within 6, 5, 4, or 3 nucleotides of the 5′ and/or 3′ terminus. Thesense strand need only be substantially complementary with the antisensestrand to maintain the overall double-strand character of the molecule.

As used herein, “modified dsRNA” refers to a dsRNA molecule thatcomprises at least one alteration that renders it more resistant tonucleases (e.g., protein kinase) than an identical dsRNA molecule thatrecognizes the same target RNA. Modified dsRNAs may include asingle-stranded nucleotide overhang and/or at least one substitutednucleotide.

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure when a3′-end of one RNA strand extends beyond the 5′-end of the othercomplementary strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that is doublestranded over its entire length, i.e., no nucleotide overhang at eitherend of the molecule.

The term “terminal base pair,” as used herein, refers to the lastnucleotide base pair on one end of the duplex region of adouble-stranded molecule. For example, if a dsRNA or other molecule isblunt ended (i.e., has no nucleotide overhangs), the last nucleotidebase pairs at both ends of the molecule are terminal base pairs. Where adsRNA or other molecule has a nucleotide overhang at one or both ends ofthe duplex structure, the last nucleotide base pair(s) immediatelyadjacent the nucleotide overhang(s) is the terminal base pair at thatend(s) of the molecule.

In certain embodiments, the methods provided herein may utilizedouble-stranded ribonucleic acid (dsRNA) molecules as modulating agents,for reducing expression of an AARS transcript such as a selectedfragment or splice variant. dsRNAs generally comprise two singlestrands. One strand of the dsRNA comprises a nucleotide sequence that issubstantially identical to a portion of the target gene or target region(the “sense” strand), and the other strand (the “complementary” or“antisense” strand) comprises a sequence that is substantiallycomplementary to a portion of the target region. The strands aresufficiently complementary to hybridize to form a duplex structure. Incertain embodiments, the complementary RNA strand may be less than 30nucleotides, less than 25 nucleotides in length, or even 19 to 24nucleotides in length. In certain aspects, the complementary nucleotidesequence may be 20-23 nucleotides in length, or 22 nucleotides inlength.

In certain embodiments, at least one of the RNA strands comprises anucleotide overhang of 1 to 4 nucleotides in length. In otherembodiments, the dsRNA may further comprise at least one chemicallymodified nucleotide. In certain aspects, a dsRNA comprising asingle-stranded overhang of 1 to 4 nucleotides may comprise a moleculewherein the unpaired nucleotide of the single-stranded overhang that isdirectly adjacent to the terminal nucleotide pair contains a purinebase. In other aspects, the last complementary nucleotide pairs on bothends of a dsRNA are a G-C pair, or, at least two of the last fourterminal nucleotide pairs are G-C pairs.

Certain embodiments of the present invention may comprise microRNAs.Micro-RNAs represent a large group of small RNAs produced naturally inorganisms, some of which regulate the expression of target genes.Micro-RNAs are formed from an approximately 70 nucleotidesingle-stranded hairpin precursor transcript by Dicer. (V. Ambros et al.Current Biology 13:807, 2003). Certain micro-RNAs may be transcribed ashairpin RNA precursors, which are then processed to their mature formsby Dicer enzyme.

Certain embodiments may also employ short-interfering RNAs (siRNA). Incertain embodiments, the first strand of the double-strandedoligonucleotide contains two more nucleoside residues than the secondstrand. In other embodiments, the first strand and the second strandhave the same number of nucleosides; however, the first and secondstrands may be offset such that the two terminal nucleosides on thefirst and second strands are not paired with a residue on thecomplimentary strand. In certain instances, the two nucleosides that arenot paired are thymidine resides.

Also included are short hairpin RNAs (shRNAs) and micro RNAs (miRNAs). Adouble-stranded structure of an shRNA is formed by a singleself-complementary RNA strand, and RNA duplex formation may be initiatedeither inside or outside the cell. MicroRNAs (miRNAs) are smallnon-coding RNAs of 20-22 nucleotides, typically excised from ˜70nucleotide foldback RNA precursor structures known as pre-miRNAs.

In instances when the modulating agent comprises siRNA, the agent shouldinclude a region of sufficient homology to the target region, and be ofsufficient length in terms of nucleotides, such that the siRNA agent, ora fragment thereof, can mediate down regulation of the target RNA. Itwill be understood that the term “ribonucleotide” or “nucleotide” can,in the case of a modified RNA or nucleotide surrogate, also refer to amodified nucleotide, or surrogate replacement moiety at one or morepositions. Thus, an siRNA agent is or includes a region which is atleast partially complementary to the target RNA, as described herein.

In addition, an siRNA modulating agent may be modified or includenucleoside surrogates. Single stranded regions of an siRNA agent may bemodified or include nucleoside surrogates, e.g., the unpaired region orregions of a hairpin structure, e.g., a region which links twocomplementary regions, can have modifications or nucleoside surrogates.Modification to stabilize one or more 3′- or 5′-terminus of an siRNAagent, e.g., against exonucleases, or to favor the antisense siRNA agentto enter into RISC are also useful. Modifications can include C3 (or C6,C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidicspacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethyleneglycol), special biotin or fluorescein reagents that come asphosphoramidites and that have another DMT-protected hydroxyl group,allowing multiple couplings during RNA synthesis.

siRNA agents may include, for example, molecules that are long enough totrigger the interferon response (which can be cleaved by Dicer(Bernstein et al. 2001. Nature, 409:363-366) and enter a RISC(RNAi-induced silencing complex)), in addition to molecules which aresufficiently short that they do not trigger the interferon response(which molecules can also be cleaved by Dicer and/or enter a RISC),e.g., molecules which are of a size which allows entry into a RISC,e.g., molecules which resemble Dicer-cleavage products. An siRNAmodulating agent, or a cleavage product thereof, can down regulate atarget gene, e.g., by inducing RNAi with respect to a target RNA,preferably an AARS target such as a selected splice variant.

Each strand of an siRNA agent can be equal to or less than 35, 30, 25,24, 23, 22, 21, 20, 19, 18, 17, 16, or 15 nucleotides in length. Thestrand is preferably at least 19 nucleotides in length. For example,each strand can be between 21 and 25 nucleotides in length. PreferredsiRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or25 nucleotide pairs, and one or more overhangs, preferably one or two 3′overhangs, of 2-3 nucleotides.

In addition to homology to target RNA and the ability to down regulate atarget gene, an siRNA agent may have one or more of the followingproperties: it may, despite modifications, even to a very large number,or all of the nucleosides, have an antisense strand that can presentbases (or modified bases) in the proper three dimensional framework soas to be able to form correct base pairing and form a duplex structurewith a homologous target RNA which is sufficient to allow downregulation of the target, e.g., by cleavage of the target RNA; it may,despite modifications, even to a very large number, or all of thenucleosides, still have “RNA-like” properties, i.e., it may possess theoverall structural, chemical and physical properties of an RNA molecule,even though not exclusively, or even partly, of ribonucleotide-basedcontent. For example, an siRNA agent can contain, e.g., a sense and/oran antisense strand in which all of the nucleotide sugars contain e.g.,2′ fluoro in place of 2′ hydroxyl. This deoxyribonucleotide-containingagent can still be expected to exhibit RNA-like properties. While notwishing to be bound by theory, the electronegative fluorine prefers anaxial orientation when attached to the C2′ position of ribose. Thisspatial preference of fluorine can, in turn, force the sugars to adopt aC₃′-endo pucker. This is the same puckering mode as observed in RNAmolecules and gives rise to the RNA-characteristic A-family-type helix.Further, since fluorine is a good hydrogen bond acceptor, it canparticipate in the same hydrogen bonding interactions with watermolecules that are known to stabilize RNA structures. Generally, it ispreferred that a modified moiety at the 2′ sugar position will be ableto enter into H-bonding which is more characteristic of the OH moiety ofa ribonucleotide than the H moiety of a deoxyribonucleotide.

A “single strand RNAi agent” as used herein, is an RNAi agent which ismade up of a single molecule. It may include a duplexed region, formedby intra-strand pairing, e.g., it may be, or include, a hairpin orpan-handle structure. Single strand RNAi modulating agents arepreferably antisense with regard to the target molecule. A single strandRNAi agent should be sufficiently long that it can enter the RISC andparticipate in RISC mediated cleavage of a target mRNA. A single strandRNAi agent is at least 14, and more preferably at least 15, 20, 25, 29,35, 40, or 50 nucleotides in length. It is preferably less than 200,100, or 60 nucleotides in length.

Hairpin RNAi modulating agents may have a duplex region equal to or atleast 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplexregion may preferably be equal to or less than 200, 100, or 50, inlength. Certain ranges for the duplex region are 15-30, 17 to 23, 19 to23, and 19 to 21 nucleotides pairs in length. The hairpin may have asingle strand overhang or terminal unpaired region, preferably the 3′,and preferably of the antisense side of the hairpin. In certainembodiments, overhangs are 2-3 nucleotides in length.

Certain modulating agents utilized according to the methods providedherein may comprise RNAi oligonucleotides such as chimericoligonucleotides, or “chimeras,” which contain two or more chemicallydistinct regions, each made up of at least one monomer unit, i.e., anucleotide in the case of an oligonucleotide compound. Theseoligonucleotides typically contain at least one region wherein theoligonucleotide is modified so as to confer upon the oligonucleotideincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid.Consequently, comparable results can often be obtained with shorteroligonucleotides when chimeric oligonucleotides are used, compared tophosphorothioate oligodeoxynucleotides. Chimeric oligonucleotides may beformed as composite structures of two or more oligonucleotides, modifiedoligonucleotides, oligonucleotides and/or oligonucleotide mimetics asdescribed above. Such oligonucleotides have also been referred to in theart as hybrids or gapmers. Representative United States patents thatteach the preparation of such hybrid structures include, but are notlimited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775;5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355;5,652,356; 5,700,922; and 5,955,589, each of which is hereinincorporated by reference. In certain embodiments, the chimericoligonucleotide is RNA-DNA, DNA-RNA, RNA-DNA-RNA, DNA-RNA-DNA, orRNA-DNA-RNA-DNA, wherein the oligonucleotide is between 5 and 60nucleotides in length.

In one aspect of the invention RNAi agents relate to an oligonucleotidecomprising at least one ligand tethered to an altered or non-naturalnucleobase. A large number of compounds can function as the alteredbase. The structure of the altered base is important to the extent thatthe altered base should not substantially prevent binding of theoligonucleotide to its target, e.g., mRNA. In certain embodiments, thealtered base is difluorotolyl, nitropyrrolyl, nitroimidazolyl,nitroindolyl, napthalenyl, anthrancenyl, pyridinyl, quinolinyl, pyrenyl,or the divalent radical of any one of the non-natural nucleobasesdescribed herein. In certain embodiments, the non-natural nucleobase isdifluorotolyl, nitropyrrolyl, or nitroimidazolyl. In certainembodiments, the non-natural nucleobase is difluorotolyl. A wide varietyof ligands are known in the art and are amenable to the presentinvention. For example, the ligand can be a steroid, bile acid, lipid,folic acid, pyridoxal, B12, riboflavin, biotin, aromatic compound,polycyclic compound, crown ether, intercalator, cleaver molecule,protein-binding agent, or carbohydrate. In certain embodiments, theligand is a steroid or aromatic compound. In certain instances, theligand is cholesteryl.

In other embodiments, the RNAi agent is an oligonucleotide tethered to aligand for the purposes of improving cellular targeting and uptake. Forexample, an RNAi agent may be tethered to an antibody, or antigenbinding fragment thereof. As an additional example, an RNAi agent may betethered to a specific ligand binding molecule, such as a polypeptide orpolypeptide fragment that specifically binds a particular cell-surfacereceptor.

In other embodiments, the modulating agent comprises a non-naturalnucleobase, as described herein. In certain instances, the ribose sugarmoiety that naturally occurs in nucleosides is replaced with a hexosesugar. In certain aspects, the hexose sugar is an allose, altrose,glucose, mannose, gulose, idose, galactose, talose, or a derivativethereof. In a preferred embodiment, the hexose is a D-hexose. In certaininstances, the ribose sugar moiety that naturally occurs in nucleosidesis replaced with a polycyclic heteroalkyl ring or cyclohexenyl group. Incertain instances, the polycyclic heteroalkyl group is a bicyclic ringcontaining one oxygen atom in the ring. In certain instances, thepolycyclic heteroalkyl group is a bicyclo[2.2.1]heptane, abicyclo[3.2.1]octane, or a bicyclo[3.3.1]nonane. Examples of modifiedRNAi agents also include oligonucleotides containing modified backbonesor non-natural internucleoside linkages, as described herein.

The present invention further encompasses oligonucleotides employingribozymes. Synthetic RNA molecules and derivatives thereof that catalyzehighly specific endoribonuclease activities are known as ribozymes.(see, e.g., U.S. Pat. No. 5,543,508 to Haseloff et al., and U.S. Pat.No. 5,545,729 to Goodchild et al.) The cleavage reactions are catalyzedby the RNA molecules themselves. In naturally occurring RNA molecules,the sites of self-catalyzed cleavage are located within highly conservedregions of RNA secondary structure (Buzayan et al., Proc. Natl. Acad.Sci. U.S.A., 1986, 83, 8859; Forster et al., Cell, 1987, 50, 9).Naturally occurring autocatalytic RNA molecules have been modified togenerate ribozymes which can be targeted to a particular cellular orpathogenic RNA molecule with a high degree of specificity. Thus,ribozymes serve the same general purpose as antisense oligonucleotides(i.e., modulation of expression of a specific gene) and, likeoligonucleotides, are nucleic acids possessing significant portions ofsingle-strandedness.

In certain instances, the RNAi agents or antisense oligonucleotides foruse with the methods provided herein may be modified by non-ligandgroup. A number of non-ligand molecules have been conjugated tooligonucleotides in order to enhance the activity, cellulardistribution, cellular targeting, or cellular uptake of theoligonucleotide, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Letsinger et al., Proc.Natl. Acad. Sci. USA, 1989, 86:6553), arginine-rich peptides, cholicacid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), athioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993,3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992,20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues(Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBSLett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), aphospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such oligonucleotideconjugates have been listed above. Typical conjugation protocols involvethe synthesis of oligonucleotides bearing an aminolinker at one or morepositions of the sequence. The amino group is then reacted with themolecule being conjugated using appropriate coupling or activatingreagents. The conjugation reaction may be performed either with theoligonucleotide still bound to the solid support or following cleavageof the oligonucleotide in solution phase. Purification of theoligonucleotide conjugate by HPLC typically affords the pure conjugate.

Additional examples of RNAi agents may be found in U.S. ApplicationPublication Nos. 2007/0275465, 2007/0054279, 2006/0287260, 2006/0035254,2006/0008822, which are incorporated by reference. Also included arevector delivery systems that are capable of expressing theAARS-targeting sequences described herein. Included are vectors thatexpress siRNA or other duplex-forming RNA interference molecules.

A vector or nucleic acid construct system can comprise a single vectoror plasmid, two or more vectors or plasmids, which together contain thetotal DNA to be introduced into the genome of the host cell, or atransposon. The choice of the vector will typically depend on thecompatibility of the vector with the host cell into which the vector isto be introduced. In the present case, the vector or nucleic acidconstruct is preferably one which is operably functional in a mammaliancell, such as a muscle cell. The vector can also include a selectionmarker such as an antibiotic or drug resistance gene, or a reporter gene(i.e., green fluorescent protein, luciferase), that can be used forselection or identification of suitable transformants or transfectants.Exemplary delivery systems may include viral vector systems (i.e.,viral-mediated transduction) including, but not limited to, retroviral(e.g., lentiviral) vectors, adenoviral vectors, adeno-associated viralvectors, and herpes viral vectors, among others known in the art.

XI. DRUG DISCOVERY

Certain embodiments relate to the use of AARS polypeptides, antibodies,or polynucleotides in drug discovery, typically to identify agents thatmodulate one or more of the non-canonical activities of the referenceAARS polypeptide, e.g., the AARS protein fragment. For example, certainembodiments include methods of identifying one or more “cellular bindingpartners” of an AARS reference polypeptide, such as a cellular protein,lipid, nucleic acid or other host molecule that directly or physicallyinteracts with the AARS polypeptide. Particular examples include forexample cell-surface receptors, such as GPCRs, protein-proteininteraction domains, and extracellular or intracellular domains thereof.

Also included are methods of identifying host molecules that participatein one or more non-canonical activities of the AARS polypeptide,including molecules that directly or indirectly interact with thecellular binding partner, and either regulate its role in anon-canonical activity, or are regulated by the binding partner. Suchhost molecules include both upstream and downstream components of thenon-canonical pathway, typically related by about 1, 2, 3, 4, 5 or moreidentifiable steps in the pathway, relative to the cellular bindingpartner/AARS protein interaction.

Certain aspects include methods of identifying a compound (e.g.,polypeptide) or other agent that agonizes or antagonizes thenon-canonical activity of an AARS reference polypeptide or activevariant thereof, such as by interacting with the AARS polypeptide and/orone or more of its cellular binding partners. Also included are methodsof identifying agents that modulate the expression (e.g., splicing) ofAARS splice variants, or modulate the activity of proteases thatotherwise regulate the production of endogenous AARS protein fragments(resectins) at the protein level.

Certain embodiments therefore include methods of identifying a bindingpartner of an AARS reference polypeptide, comprising a) combining theAARS polypeptide with a biological sample under suitable conditions, andb) detecting specific binding of the AARS polypeptide to a bindingpartner, thereby identifying a binding partner that specifically bindsto the AARS reference polypeptide. Also included are methods ofscreening for a compound that specifically binds to an AARS referencepolypeptide or a binding partner of the AARS polypeptide, comprising a)combining the polypeptide or the binding partner with at least one testcompound under suitable conditions, and b) detecting binding of thepolypeptide or the binding partner to the test compound, therebyidentifying a compound that specifically binds to the polypeptide or itsbinding partner. In certain embodiments, the compound is a polypeptideor peptide. In certain embodiments, the compound is a small molecule orother (e.g., non-biological) chemical compound. In certain embodiments,the compound is a peptide mimetic.

Any method suitable for detecting protein-protein interactions may beemployed for identifying cellular proteins that interact with an AARSreference polypeptide, interact with one or more of its cellular bindingpartners, or both. Examples of traditional methods that may be employedinclude co-immunoprecipitation, cross-linking, and co-purificationthrough gradients or chromatographic columns of cell lysates or proteinsobtained from cell lysates, mainly to identify proteins in the lysatethat interact with the AARS polypeptide.

In these and related embodiments, at least a portion of the amino acidsequence of a protein that interacts with an AARS polypeptide or itsbinding partner can be ascertained using techniques well known to thoseof skill in the art, such as via the Edman degradation technique. See,e.g., Creighton Proteins: Structures and Molecular Principles, W. H.Freeman & Co., N.Y., pp. 34 49, 1983. The amino acid sequence obtainedmay be used as a guide for the generation of oligonucleotide mixturesthat can be used to screen for gene sequences encoding such proteins.Screening may be accomplished, for example, by standard hybridization orPCR techniques, as described herein and known in the art. Techniques forthe generation of oligonucleotide mixtures and the screening are wellknown. See, e.g., Ausubel et al. Current Protocols in Molecular BiologyGreen Publishing Associates and Wiley Interscience, N.Y., 1989; andInnis et al., eds. PCR Protocols: A Guide to Methods and ApplicationsAcademic Press, Inc., New York, 1990.

Additionally, methods may be employed in the simultaneous identificationof genes that encode the binding partner or other polypeptide. Thesemethods include, for example, probing expression libraries, in a mannersimilar to the well known technique of antibody probing of lambda-gt11libraries, using labeled AARS protein, or another polypeptide, peptideor fusion protein, e.g., a variant AARS polypeptide or AARS domain fusedto a marker (e.g., an enzyme, fluor, luminescent protein, or dye), or anIg-Fc domain.

One method that detects protein interactions in vivo, the two-hybridsystem, is described in detail for illustration only and not by way oflimitation. One example of this system has been described (Chien et al.,PNAS USA 88:9578 9582, 1991) and is commercially available from Clontech(Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids may be constructed thatencode two hybrid proteins: one plasmid consists of nucleotides encodingthe DNA-binding domain of a transcription activator protein fused to anAARS reference nucleotide sequence (or, in certain embodiments, itsbinding partner), or a variant thereof, and the other plasmid consistsof nucleotides encoding the transcription activator protein's activationdomain fused to a cDNA (or collection of cDNAs) encoding an unknownprotein(s) that has been recombined into the plasmid as part of a cDNAlibrary. The DNA-binding domain fusion plasmid and the activator cDNAlibrary may be transformed into a strain of the yeast Saccharomycescerevisiae that contains a reporter gene (e.g., HBS or lacZ) whoseregulatory region contains the transcription activator's binding site.Either hybrid protein alone cannot activate transcription of thereporter gene: the DNA-binding domain hybrid cannot because it does notprovide activation function and the activation domain hybrid cannotbecause it cannot localize to the activator's binding sites. Interactionof the two hybrid proteins reconstitutes the functional activatorprotein and results in expression of the reporter gene, which isdetected by an assay for the reporter gene product.

The two-hybrid system or other such methodology may be used to screenactivation domain libraries for proteins that interact with the “bait”gene product. By way of example, and not by way of limitation, an AARSreference polypeptide or variant may be used as the bait gene product.An AARS binding partner may also be used as a “bait” gene product. Totalgenomic or cDNA sequences are fused to the DNA encoding an activationdomain. This library and a plasmid encoding a hybrid of a bait AARS geneproduct fused to the DNA-binding domain are co-transformed into a yeastreporter strain, and the resulting transformants are screened for thosethat express the reporter gene.

A cDNA library of the cell line from which proteins that interact withbait AARS gene products are to be detected can be made using methodsroutinely practiced in the art. For example, the cDNA fragments can beinserted into a vector such that they are translationally fused to thetranscriptional activation domain of GAL4. This library can beco-transformed along with the bait gene-GAL4 fusion plasmid into a yeaststrain, which contains a lacZ gene driven by a promoter that containsGAL4 activation sequence. A cDNA encoded protein, fused to GAL4transcriptional activation domain, that interacts with bait gene productwill reconstitute an active GAL4 protein and thereby drive expression ofthe HIS3 gene. Colonies, which express HIS3, can be detected by theirgrowth on Petri dishes containing semi-solid agar based media lackinghistidine. The cDNA can then be purified from these strains, and used toproduce and isolate the bait AARS gene-interacting protein usingtechniques routinely practiced in the art.

Also included are three-hybrid systems, which allow the detection ofRNA-protein interactions in yeast. See, e.g., Hook et al., RNA.11:227-233, 2005. Accordingly, these and related methods can be used toidentify a cellular binding partner of an AARS polypeptide, and toidentify other proteins or nucleic acids that interact with the AARSpolypeptide, the cellular binding partner, or both.

Certain embodiments relate to the use of interactome screeningapproaches. Particular examples include protein domain-based screening(see, e.g., Boxem et al., Cell. 134:534-545, 2008; and Yu et al.,Science. 322:10-110, 2008).

As noted above, once isolated, binding partners can be identified andcan, in turn, be used in conjunction with standard techniques toidentify proteins or other compounds with which it interacts. Certainembodiments thus relate to methods of screening for a compound thatspecifically binds to the binding partner of an AARS referencepolypeptide, comprising a) combining the binding partner with at leastone test compound under suitable conditions, and b) detecting binding ofthe binding partner to the test compound, thereby identifying a compoundthat specifically binds to the binding partner. In certain embodiments,the test compound is a polypeptide. In certain embodiments, the testcompound is a chemical compound, such as a small molecule compound orpeptide mimetic.

Certain embodiments include methods of screening for a compound thatmodulates the activity of an AARS reference polypeptide, comprising a)combining the polypeptide with at least one test compound underconditions permissive for the activity of the polypeptide, b) assessingthe activity of the polypeptide in the presence of the test compound,and c) comparing the activity of the polypeptide in the presence of thetest compound with the activity of the polypeptide in the absence of thetest compound, wherein a change in the activity of the polypeptide inthe presence of the test compound is indicative of a compound thatmodulates the activity of the polypeptide. Certain embodiments includemethods of screening for a compound that modulates the activity of abinding partner of an AARS reference polypeptide, comprising a)combining the polypeptide with at least one test compound underconditions permissive for the activity of the binding partner, b)assessing the activity of the binding partner in the presence of thetest compound, and c) comparing the activity of the binding partner inthe presence of the test compound with the activity of the bindingpartner in the absence of the test compound, wherein a change in theactivity of the binding partner in the presence of the test compound isindicative of a compound that modulates the activity of the bindingpartner. Typically, these and related embodiments include assessing aselected non-canonical activity that is associated with the AARSpolypeptide or its binding partner. Included are in vitro and in vivoconditions, such as cell culture conditions.

Certain embodiments include methods of screening a compound foreffectiveness as a full or partial agonist of an AARS referencepolypeptide or an active fragment or variant thereof, comprising a)exposing a sample comprising the polypeptide to a compound, and b)detecting agonist activity in the sample, typically by measuring anincrease in the non-canonical activity of the AARS polypeptide. Certainmethods include a) exposing a sample comprising a binding partner of theAARS polypeptide to a compound, and b) detecting agonist activity in thesample, typically by measuring an increase in the selected non-canonicalactivity of the AARS polypeptide. Certain embodiments includecompositions that comprise an agonist compound identified by the methodand a pharmaceutically acceptable carrier or excipient.

Also included are methods of screening a compound for effectiveness as afull or partial antagonist of an AARS reference polypeptide, comprisinga) exposing a sample comprising the polypeptide to a compound, and b)detecting antagonist activity in the sample, typically by measuring adecrease in the non-canonical activity of the AARS polypeptide. Certainmethods include a) exposing a sample comprising a binding partner of theAARS polypeptide to a compound, and b) detecting antagonist activity inthe sample, typically by measuring a decrease in the selectednon-canonical activity of the AARS polypeptide. Certain embodimentsinclude compositions that comprise an antagonist compound identified bythe method and a pharmaceutically acceptable carrier or excipient.

In certain embodiments, in vitro systems may be designed to identifycompounds capable of interacting with or modulating an AARS referencesequence or its binding partner. Certain of the compounds identified bysuch systems may be useful, for example, in modulating the activity ofthe pathway, and in elaborating components of the pathway itself. Theymay also be used in screens for identifying compounds that disruptinteractions between components of the pathway; or may disrupt suchinteractions directly. One exemplary approach involves preparing areaction mixture of the AARS polypeptide and a test compound underconditions and for a time sufficient to allow the two to interact andbind, thus forming a complex that can be removed from and/or detected inthe reaction mixture

In vitro screening assays can be conducted in a variety of ways. Forexample, an AARS polypeptide, a cellular binding partner, or testcompound(s) can be anchored onto a solid phase. In these and relatedembodiments, the resulting complexes may be captured and detected on thesolid phase at the end of the reaction. In one example of such a method,the AARS polypeptide and/or its binding partner are anchored onto asolid surface, and the test compound(s), which are not anchored, may belabeled, either directly or indirectly, so that their capture by thecomponent on the solid surface can be detected. In other examples, thetest compound(s) are anchored to the solid surface, and the AARSpolypeptide and/or its binding partner, which are not anchored, arelabeled or in some way detectable. In certain embodiments, microtiterplates may conveniently be utilized as the solid phase. The anchoredcomponent (or test compound) may be immobilized by non-covalent orcovalent attachments. Non-covalent attachment may be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized may be used toanchor the protein to the solid surface. The surfaces may be prepared inadvance and stored.

To conduct an exemplary assay, the non-immobilized component istypically added to the coated surface containing the anchored component.After the reaction is complete, un-reacted components are removed (e.g.,by washing) under conditions such that any specific complexes formedwill remain immobilized on the solid surface. The detection of complexesanchored on the solid surface can be accomplished in a number of ways.For instance, where the previously non-immobilized component ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the previously non-immobilizedcomponent is not pre-labeled, an indirect label can be used to detectcomplexes anchored on the surface; e.g., using a labeled antibodyspecific for the previously non-immobilized component (the antibody, inturn, may be directly labeled or indirectly labeled with a labeledanti-Ig antibody).

Alternatively, the presence or absence of binding of a test compound canbe determined, for example, using surface plasmon resonance (SPR) andthe change in the resonance angle as an index, wherein an AARSpolypeptide or a cellular binding partner is immobilized onto thesurface of a commercially available sensorchip (e.g., manufactured byBIACORE™) according to a conventional method, the test compound iscontacted therewith, and the sensorchip is illuminated with a light of aparticular wavelength from a particular angle. The binding of a testcompound can also be measured by detecting the appearance of a peakcorresponding to the test compound by a method wherein an AARSpolypeptide or a cellular binding partner is immobilized onto thesurface of a protein chip adaptable to a mass spectrometer, a testcompound is contacted therewith, and an ionization method such asMALDI-MS, ESI-MS, FAB-MS and the like is combined with a massspectrometer (e.g., double-focusing mass spectrometer, quadrupole massspectrometer, time-of-flight mass spectrometer, Fourier transformationmass spectrometer, ion cyclotron mass spectrometer and the like).

In certain embodiments, cell-based assays, membrane vesicle-basedassays, or membrane fraction-based assays can be used to identifycompounds that modulate interactions in the non-canonical pathway of theselected AARS polypeptide. To this end, cell lines that express an AARSpolypeptide and/or a binding partner, or a fusion protein containing adomain or fragment of such proteins (or a combination thereof), or celllines (e.g., COS cells, CHO cells, HEK293 cells, Hela cells etc.) thathave been genetically engineered to express such protein(s) or fusionprotein(s) can be used. Test compound(s) that influence thenon-canonical activity can be identified by monitoring a change (e.g., astatistically significant change) in that activity as compared to acontrol or a predetermined amount.

For embodiments that relate to antisense and RNAi agents, for example,also included are methods of screening a compound for effectiveness inaltering expression of an AARS reference polynucleotide, comprising a)exposing a sample comprising the AARS reference polynucleotide to acompound such as a potential antisense oligonucleotide, and b) detectingaltered expression of the AARS polynucleotide. In certain non-limitingexamples, these and related embodiments can be employed in cell-basedassays or in cell-free translation assays, according to routinetechniques in the art. Also included are the antisense and RNAi agentsidentified by such methods.

Antibodies to AARS protein fragments can also be used in screeningassays, such as to identify an agent that specifically binds to an AARS,confirm the specificity or affinity of an agent that binds to an AARSprotein fragment, or identify the site of interaction between the agentand the AARS protein fragment. Included are assays in which the antibodyis used as a competitive inhibitor of the agent. For instance, anantibody that specifically binds to an AARS protein fragment with aknown affinity can act as a competitive inhibitor of a selected agent,and be used to calculate the affinity of the agent for the AARS proteinfragment. Also, one or more antibodies that specifically bind to knownepitopes or sites of an AARS protein fragment can be used as acompetitive inhibitor to confirm whether or not the agent binds at thatsame site. Other variations will be apparent to persons skilled in theart.

Also included are any of the above methods, or other screening methodsknown in the art, which are adapted for high-throughput screening (HTS).HTS typically uses automation to run a screen of an assay against alibrary of candidate compounds, for instance, an assay that measures anincrease or a decrease in a non-canonical activity, as described herein.

Any of the screening methods provided herein may utilize small moleculelibraries or libraries generated by combinatorial chemistry. Librariesof chemical and/or biological mixtures, such as fungal, bacterial, oralgal extracts, are known in the art and can be screened with any of theassays of the invention. Examples of methods for the synthesis ofmolecular libraries can be found in: (Carell et al., 1994a; Carell etal., 1994b; Cho et al., 1993; DeWitt et al., 1993; Gallop et al., 1994;Zuckermann et al., 1994).

Libraries of compounds may be presented in solution (Houghten et al.,1992) or on beads (Lam et al., 1991), on chips (Fodor et al., 1993),bacteria, spores (Ladner et al., U.S. Pat. No. 5,223,409, 1993),plasmids (Cull et al., 1992) or on phage (Cwirla et al., 1990; Devlin etal., 1990; Felici et al., 1991; Ladner et al., U.S. Pat. No. 5,223,409,1993; Scott and Smith, 1990). Embodiments of the present inventionencompass the use of different libraries for the identification of smallmolecule modulators of one or more AARS protein fragments, theircellular binding partners, and/or their related non-canonicalactivities. Libraries useful for the purposes of the invention include,but are not limited to, (1) chemical libraries, (2) natural productlibraries, and (3) combinatorial libraries comprised of random peptides,oligonucleotides and/or organic molecules.

Chemical libraries consist of structural analogs of known compounds orcompounds that are identified as “hits” or “leads” via natural productscreening. Natural product libraries are derived from collections ofmicroorganisms, animals, plants, or marine organisms which are used tocreate mixtures for screening by: (1) fermentation and extraction ofbroths from soil, plant or marine microorganisms or (2) extraction ofplants or marine organisms. Natural product libraries includepolyketides, non-ribosomal peptides, and variants (non-naturallyoccurring) thereof. See, e.g., Cane et al., Science 282:63-68, 1998.Combinatorial libraries may be composed of large numbers of peptides,oligonucleotides or organic compounds as a mixture. They are relativelyeasy to prepare by traditional automated synthesis methods, PCR, cloningor proprietary synthetic methods.

More specifically, a combinatorial chemical library is a collection ofdiverse chemical compounds generated by either chemical synthesis orbiological synthesis, by combining a number of chemical “buildingblocks” such as reagents. For example, a linear combinatorial chemicallibrary such as a polypeptide library is formed by combining a set ofchemical building blocks (amino acids) in every possible way for a givencompound length (i.e., the number of amino acids in a polypeptidecompound). Millions of chemical compounds can be synthesized throughsuch combinatorial mixing of chemical building blocks.

For a review of combinatorial chemistry and libraries created therefrom,see, e.g., Huc, I. and Nguyen, R. (2001) Comb. Chem. High ThroughputScreen 4:53-74; Lepre, C A. (2001) Drug Discov. Today 6:133-140; Peng,S. X. (2000) Biomed. Chromatogr. 14:430-441; Bohm, H. J. and Stahl, M.(2000) Curr. Opin. Chem. Biol. 4:283-286; Barnes, C and Balasubramanian,S. (2000) Curr. Opin. Chem. Biol. 4:346-350; Lepre, Enjalbal, C, et al.,(2000) Mass Septrom Rev. 19:139-161; Hall, D. G., (2000) Nat.Biotechnol. 18:262-262; Lazo, J. S., and Wipf, P. (2000) J. Pharmacol.Exp. Ther. 293:705-709; Houghten, R. A., (2000) Ann. Rev. Pharmacol.Toxicol. 40:273-282; Kobayashi, S. (2000) Curr. Opin. Chem. Biol. (2000)4:338-345; Kopylov, A. M. and Spiridonova, V. A. (2000) Mol. Biol.(Mosk) 34:1097-1113; Weber, L. (2000) Curr. Opin. Chem. Biol. 4:295-302;Dolle, R. E. (2000) J. Comb. Chem. 2:383-433; Floyd, C D., et al.,(1999) Prog. Med. Chem. 36:91-168; Kundu, B., et al., (1999) Prog. DrugRes. 53:89-156; Cabilly, S. (1999) Mol. Biotechnol. 12:143-148; Lowe, G.(1999) Nat. Prod. Rep. 16:641-651; Dolle, R. E. and Nelson, K. H. (1999)J. Comb. Chem. 1:235-282; Czarnick, A. W. and Keene, J. D. (1998) Curr.Biol. 8:R705-R707; Dolle, R. E. (1998) Mol. Divers. 4:233-256; Myers, P.L., (1997) Curr. Opin. Biotechnol. 8:701-707; and Pluckthun, A. andCortese, R. (1997) Biol. Chem. 378:443.

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc.,St. Louis, Mo., ChemStar, Ltd., Moscow, RU, 3D Pharmaceuticals, Exton,Pa., Martek Biosciences, Columbia, Md., etc.).

XII. METHODS OF USE

Embodiments of the present invention include therapeutic methods oftreatment. Accordingly, the AARS agents described herein, including AARSpolypeptides, AARS polynucleotides, AARS polynucleotide-based vectors,AARS expressing host cells, antisense oligonucleotides, RNAi agents, aswell as binding agents such as peptides, antibodies and antigen-bindingfragments, peptide mimetics and other small molecules, can be used totreat a variety of non-limiting diseases or conditions associated withthe non-canonical activities of a reference AARS. Examples of suchnon-canonical activities include modulation of extracellular signaling,modulation of cell proliferation, modulation of cell migration,modulation of cell differentiation (e.g., hematopoiesis, neurogenesis,myogenesis, osteogenesis, and adipogenesis), modulation of apoptosis orother forms of cell death, modulation of angiogenesis, modulation ofcell binding, modulation of cellular metabolism, modulation of cytokineproduction or activity, modulation of cytokine receptor activity,modulation of cellular uptake, or secretion, immunomodulation,modulation of inflammation, modulation of metabolic processes such asglucose control, and the like.

Included are polynucleotide-based therapies, such as antisense therapiesand RNAi interference therapies, which typically relate to reducing theexpression of a target molecule, such as an endogenous fragment of anAARS, or a cellular binding partner of an AARS polypeptide, whichotherwise contributes to its non-canonical activity. Antisense or RNAitherapies typically antagonize the non-canonical activity, such as byreducing expression of the AARS reference polypeptide. Also included arepolypeptides or peptides, antibodies or antigen-binding fragment,peptide mimetics, or other small molecule-based therapies, which eitheragonize or antagonize the non-canonical activity of an AARS referencepolypeptide, such as by interacting directly with the AARS polypeptide,its cellular binding partner(s), or both.

These and related embodiments include methods of using the AARS agentsor compositions of the present invention for treating a cell, tissue orsubject. The cells or tissues that may be treated or modulated by thepresent invention are preferably mammalian cells or tissues, or morepreferably human cells or tissues. Such cells or tissues can be of ahealthy state or of a diseased state.

In certain embodiments, for example, methods are provided for modulatingtherapeutically relevant cellular activities including, but not limitedto, cellular metabolism, cell differentiation, cell proliferation,cellular uptake, cell secretion, cell death, cell mobilization, cellmigration, gene transcription, mRNA translation, cell impedance, immuneresponses, inflammatory responses, and the like, comprising contacting acell with an AARS agent or composition as described herein. In certainembodiments, the cell is in a subject. Accordingly, the AARScompositions may be employed in treating essentially any cell or tissueor subject that would benefit from modulation of one or more suchactivities.

The AARS agents and compositions may also be used in any of a number oftherapeutic contexts including, for example, those relating to thetreatment or prevention of neoplastic diseases, immune system diseasesor conditions (e.g., autoimmune diseases and inflammation), infectiousdiseases, metabolic diseases, neuronal/neurological diseases,muscular/cardiovascular diseases, diseases associated with aberranthematopoiesis, diseases associated with aberrant myogenesis, diseasesassociated with aberrant neurogenesis, diseases associated with aberrantadipogenesis, diseases associated with aberrant osteogenesis, diseasesassociated with aberrant angiogenesis, diseases associated with aberrantcell survival, diseases associated with aberrant lipid uptake, diseasesassociated with aging (e.g. hearing loss, peripheral or autonomicneuropathies, senile dementia, retinopathy) and others.

For example, in certain illustrative embodiments, the AARS compositionsof the invention may be used to modulate angiogenesis, e.g., viamodulation of endothelial cell proliferation and/or signaling.Endothelial cell proliferation and/or signaling may be monitored usingan appropriate cell line (e.g., human microvascular endothelial lungcells (HMVEC-L) and human umbilical vein endothelial cells (HUVEC)), andusing an appropriate assay (e.g., endothelial cell migration assays,endothelial cell proliferation assays, tube-forming assays, matrigelplug assays, etc.), many of which are known and available in the art.

Therefore, in related embodiments, the compositions of the invention maybe employed in the treatment of essentially any cell or tissue orsubject that would benefit from modulation of angiogenesis. For example,in some embodiments, a cell or tissue or subject experiencing orsusceptible to angiogenesis (e.g., an angiogenic condition) may becontacted with a suitable composition of the invention to inhibit anangiogenic condition. In other embodiments, a cell or tissueexperiencing or susceptible to insufficient angiogenesis (e.g., anangiostatic condition) may be contacted with an appropriate compositionof the invention in order to interfere with angiostatic activity and/orpromote angiogenesis.

Also included are methods of modulating hematopoiesis and relatedconditions. Examples of hematopoietic processes that may be modulated bythe AARS polypeptides of the invention include, without limitation, theformation of myeloid cells (e.g., erythroid cells, mast cellsmonocytes/macrophages, myeloid dendritic cells, granulocytes such asbasophils, neutrophils, and eosinophils, megakaryocytes, platelets) andlymphoid cells (e.g., natural killer cells, lymphoid dendritic cells,B-cells, and T-cells). Certain specific hematopoietic processes includeerythropoiesis, granulopoiesis, lymphopoiesis, megakaryopoiesis,thrombopoiesis, and others. Also included are methods of modulating thetrafficking or mobilization of hematopoietic cells, includinghematopoietic stem cells, progenitor cells, erythrocytes, granulocytes,lymphocytes, megakaryocytes, and thrombocytes.

The methods of modulating hematopoiesis may be practiced in vivo, invitro, ex vivo, or in any combination thereof. These methods can bepracticed on any biological sample, cell culture, or tissue thatcontains hematopoietic stem cells, hematopoietic progenitor cells, orother stem or progenitor cells that are capable of differentiating alongthe hematopoietic lineage (e.g., adipose tissue derived stem cells). Forin vitro and ex vivo methods, stem cells and progenitor cells, whetherof hematopoietic origin or otherwise, can be isolated and/or identifiedaccording to the techniques and characteristics described herein andknown in the art.

The compositions of the invention may also be useful as immunomodulatorsfor treating anti- or pro-inflammatory indications by modulating thecells that mediate, either directly or indirectly, autoimmune and/orinflammatory diseases, conditions and disorders. The utility of thecompositions of the invention as immunomodulators or modulators ofinflammation can be monitored using any of a number of known andavailable techniques in the art including, for example, migration assays(e.g., using leukocytes or lymphocytes) or cell viability assays (e.g.,using B-cells, T-cells, monocytes or NK cells).

“Inflammation” refers generally to the biological response of tissues toharmful stimuli, such as pathogens, damaged cells (e.g., wounds), andirritants. The term “inflammatory response” refers to the specificmechanisms by which inflammation is achieved and regulated, including,merely by way of illustration, immune cell activation or migration,cytokine production, vasodilation, including kinin release,fibrinolysis, and coagulation, among others described herein and knownin the art.

Clinical signs of chronic inflammation are dependent upon duration ofthe illness, inflammatory lesions, cause and anatomical area affected.(see, e.g., Kumar et al., Robbins Basic Pathology-8^(th) Ed., 2009Elsevier, London; Miller, L M, Pathology Lecture Notes, AtlanticVeterinary College, Charlottetown, PEI, Canada). Chronic inflammation isassociated with a variety of pathological conditions or diseases,including, for example, allergies, Alzheimer's disease, anemia, aorticvalve stenosis, arthritis such as rheumatoid arthritis andosteoarthritis, cancer, congestive heart failure, fibromyalgia,fibrosis, heart attack, kidney failure, lupus, pancreatitis, stroke,surgical complications, inflammatory lung disease, inflammatory boweldisease, atherosclerosis, neurological disorders, diabetes, metabolicdisorders, obesity, and psoriasis, among others described herein andknown in the art. Hence, AARS compositions may be used to treat ormanage chronic inflammation, modulate any of one or more of theindividual chronic inflammatory responses, or treat any one or morediseases or conditions associated with chronic inflammation.

Criteria for assessing the signs and symptoms of inflammatory and otherconditions, including for purposes of making differential diagnosis andalso for monitoring treatments such as determining whether atherapeutically effective dose has been administered in the course oftreatment, e.g., by determining improvement according to acceptedclinical criteria, will be apparent to those skilled in the art and areexemplified by the teachings of e.g., Berkow et al., eds., The MerckManual, 16^(th) edition, Merck and Co., Rahway, N.J., 1992; Goodman etal., eds., Goodman and Gilman's The Pharmacological Basis ofTherapeutics, 10^(th) edition, Pergamon Press, Inc., Elmsford, N.Y.,(2001); Avery's Drug Treatment: Principles and Practice of ClinicalPharmacology and Therapeutics, 3rd edition, ADIS Press, Ltd., Williamsand Wilkins, Baltimore, Md. (1987); Ebadi, Pharmacology, Little, Brownand Co., Boston, (1985); Osolci al., eds., Remington's PharmaceuticalSciences, 18^(th) edition, Mack Publishing Co., Easton, Pa. (1990);Katzung, Basic and Clinical Pharmacology, Appleton and Lange, Norwalk,Conn. (1992).

In other embodiments, the AARS compositions of the invention may be usedto modulate cellular proliferation and/or survival and, accordingly, fortreating or preventing diseases, disorders or conditions characterizedby abnormalities in cellular proliferation and/or survival. For example,in certain embodiments, the AARS compositions may be used to modulateapoptosis and/or to treat diseases or conditions associated withabnormal apoptosis. Apoptosis can be monitored by any of a number ofavailable techniques known and available in the art including, forexample, assays that measure fragmentation of DNA, alterations inmembrane asymmetry, activation of apoptotic caspases and/or release ofcytochrome C and AIF.

Embodiments of the present invention also include methods of treatingand preventing diseases associated with the mutation, inappropriatesubcellular association, over-expression, and/or subcellulardistribution of a parental aminoacyl-tRNA synthetase (AARS) (See, e.g.,Park et al., PNAS USA. 105:11043-11049, 2008).

Examples of diseases associated with the mutation of AARSs include, forexample, Charcot-Marie-Tooth Disease and Distal Spinal Muscular AtrophyType V, caused by mutations in Glycyl tRNA synthetase, Tyrosyl tRNAsynthetase, Lysyl tRNA synthetase, and Alanyl tRNA synthetase, amongother aminoacyl-tRNA synthetases (see McLaughlin et al., Am J Hum Genet.87:560-566, 2010; Jordanova et al., Nat Genet. 38:197-202, 2006; andLatour et al., Am J Hum Genet. 86:77-82, 2010); diabetic nephropathy,associated with mutations in Cysteinyl tRNA synthetase (see Pezzolesi etal., Diabetes. 58:1403-1410, 2009); and leukoenchalopathy, associatedwith mutations in mitochrondrial Aspartyl tRNA synthetase. Examples ofdiseases related to inappropriate subcellular association ofaminoacyl-tRNA synthetases include, for example, amyotrophic lateralsclerosis; associated with the interaction between Lysyl tRNA synthetaseand superoxide dismutase 1 (Banks et al., PLOSone 4(7)e6218:1-12, 2009);and Parkinson's disease, associated with the interaction between AIMP-2(p38) and Parkin (Choi et al., 7(3)e1001351:1-13, 2011). Examples ofdiseases caused by, or associated with, the over-expression of tRNAsynthetases, include for example, the association of variousaminoacyl-tRNA synthetases, such as methionyl, cysteinyl, isoleucyl,glutamyl-prolyl, phenylalanyl, glycyl, lysyl, tyrosyl and tryptophanyltRNA synthetases in cancer development and progression (see, e.g.,Kushner et al., Proc. Soc. Exp. Biol. Med. 153 273-276, 1976; Waseniuset al., Clin. Cancer. Res. 9:68-75, 2003; Scandurro et al., Int. J.Oncol. 19:129-135, 2001; Park et al., PNAS USA 102:6356-6361, 2005; andPark et al., PNAS USA 105: 11043-11049, 2008).

Thus in certain embodiments, the present invention provides soluble AARSprotein fragments that exhibit favorable protein stability andaggregation characteristics, and the ability to be expressed andproduced at high level in prokaryotic expression systems, which can beused to complement, or suppress the activity of a parental tRNAsynthetase associated with a disease.

Accordingly the present invention also includes therapeutic methods forthe use of such AARS protein fragments for the treatment and preventionof diseases associated with aminoacyl-tRNA synthetases. Without beingbound by any particular theory of operation, it is believed that suchAARS protein fragments may act to complement a lost function of a mutantaminoacyl-tRNA synthetase, suppress a change in the conformation,rigidity, alter the dimerization state of a mutant AARS, and/or act as adecoy to a second molecule which would otherwise inappropriatelyinteract with the wild type or mutant AARS.

In certain embodiments, such therapeutic methods include theadministration of one or more of the Tyrosyl AARS protein fragments ofTables 1-3, to a subject which has, or is at risk of developing, adisease which is characterized by the mutation, inappropriatesubcellular association, over-expression, and/or subcellulardistribution of a tyrosyl tRNA synthetase.

In certain embodiments, such AARS protein fragments may be used todevelop antibodies and binding agents which enable the development ofantibodies and binding agents to novel cryptic epitopes which may alsoact to suppress a disease phenotype associated with the parentalaminoacyl-tRNA synthetase. Accordingly, certain embodiments include theadministration of one or more antibodies or binding agents to theTyrosyl AARS polypeptides of Tables 1-3, to a subject which has, or isat risk of developing, a disease which is characterized by the mutation,inappropriate subcellular association, over-expression, and/orsubcellular distribution of a tyrosyl tRNA synthetase.

Specific examples of diseases associated with the mutation of TyrosyltRNA synthetase that can be treated with the AARS polypeptides and othercompounds of the present invention include, for example, distal spinalmuscular atrophies (dSMA) and distal hereditary motor neuropathies(dHMN), such as Charcot-Marie-Tooth Disease Type 2D (CMT2D) and DistalSpinal Muscular Atrophy Type V (dSMA-V), among others described hereinand known in the art. See, e.g., Sivakamur et al., Brain. 128:2304-2314,2005; and Antonellis et al., Am. J. Hum. Genet. 72:1293-1299, 2003.

Distal spinal muscular atrophies (dSMA) and distal hereditary motorneuropathies (dHMNs) such as CMT represent a group of inheriteddisorders that affect the peripheral nerves (i.e., the nerves outsidethe brain and spine). These diseases can be characterized by slowlyprogressive muscle weakness and atrophy in the distal parts of the limbscaused, for example, by progressive anterior horn cell degeneration,among other processes. CMT Type 1 (CMT1) is a peripheral motor andsensory demyelinating neuropathy caused, for example, by degeneration ofSchwann cells, and CMT Type 2 (CMT2) is typically characterized byprimary axonal degeneration. CMT2 is often characterized by distalmuscular atrophy, reduced compound motor action potentials, and/orreduced sensory nerve action potentials, but normal or mildly slowedmotor nerve conduction velocity.

CMT disease is also known as known also as Morbus Charcot-Marie-Tooth,Charcot-Marie-Tooth neuropathy, hereditary motor and sensory neuropathy(HMSN), hereditary motor neuropathy type V (dHMN-V), hereditary sensorand motor neuropathy (HSMN), and peroneal muscular atrophy. Currentlyincurable, this family of diseases is one of the most common inheritedneurological disorders, with an estimated 1 in 2500 individuals affected(see, e.g., Krajewski et al., Brain. 123:1516-27, 2000; and Skre, Clin.Genet. 6:98-118, 1974).

In certain embodiments, the invention includes a method of treating asubject which has, or is at risk of developing, a CMT disease such asCMT1 or CMT2 (including CMT2D), or dSMA-V, which is characterized by oneor more mutations in TyrRS, comprising the step of administering one ormore of the tyrosyl AARS protein fragments of Tables 1-3, or anantibody, or binding agent directed to any of the tyrosyl AARS proteinfragments of Tables 1-3, which treats, reduces, or reduces the risk ofdeveloping, one or more symptoms of that disease. In certainembodiments, the subject to be treated has a particular tyrosyl mutant.

Further to the characteristics described above, exemplary clinicalsymptoms of neuronal diseases such as CMT include, without limitation,foot deformity (very high arch to feet), foot drop (inability to holdfoot horizontal), loss of lower leg muscle, leading to skinny calves,numbness in the foot or leg, “slapping” gait (feet hit the floor hardwhen walking), and weakness of the hips, legs, or feet. These symptomsoften present between mid-childhood and early adulthood. Later, similarsymptoms may appear in the arms and hands, including, for example,claw-like hand deformities. Also included are complications such as aprogressive inability to walk, a progressive weakness, and injury toareas of the body that have decreased sensation. One specific symptom isreduced nerve conduction velocity.

Accordingly, the TyrRS polypeptides, antibodies, binding agents, andother compounds described herein may reduce or ameliorate any one ormore of these characteristics, symptoms or complications of neuronaldiseases such as dSMA and dHMN, including CMT disease such as CMT1 andCMT2 (including CMT2D).

Neuronal diseases such as CMT can be diagnosed according to routinetechniques in the art. For example, physical examination may be used toobserve signs such as difficulty lifting up the foot and making toe-outmovements, lack of stretch reflexes in the legs, loss of muscle controland atrophy (shrinking of the muscles) in the foot or leg, and thickenednerve bundles under the skin of the legs. Muscle biopsies and/or nervebiopsies may be used to confirm a diagnosis, and nerve conduction testscan be used to tell the difference between different forms of thedisease.

In certain methods, the subject has, or is at risk of developing acancer, which is characterized by the over-expression of TyrRS, and atherapeutic method comprises the step of administering one or more ofthe tyrosyl AARS protein fragments of Tables 1-3, or an antibody, orbinding agent directed to any of the tyrosyl AARS protein fragments ofTables 1-3, which treats or reduces the cancer, reduces the risk ofdeveloping the cancer, and/or reduces the recurrence of cancer. Inspecific embodiments, the cancer is metastatic cancer.

The progress of these and other therapies (e.g., ex vivo therapies) canbe readily monitored by conventional methods and assays and based oncriteria known to the physician or other persons of skill in the art.

XIII. PHARMACEUTICAL FORMULATIONS, ADMINISTRATION AND KITS

Embodiments of the present invention include AARS polynucleotides, AARSpolypeptides, host cells expressing AARS polypeptides, binding agents,modulatory agents, or other compounds described herein, formulated inpharmaceutically-acceptable or physiologically-acceptable solutions foradministration to a cell or an animal, either alone, or in combinationwith one or more other modalities of therapy. It will also be understoodthat, if desired, the compositions of the invention may be administeredin combination with other agents as well, such as, e.g., other proteinsor polypeptides or various pharmaceutically-active agents. There isvirtually no limit to other components that may also be included in thecompositions, provided that the additional agents do not adverselyaffect the modulatory or other effects desired to be achieved.

In the pharmaceutical compositions of the invention, formulation ofpharmaceutically-acceptable excipients and carrier solutions iswell-known to those of skill in the art, as is the development ofsuitable dosing and treatment regimens for using the particularcompositions described herein in a variety of treatment regimens,including e.g., oral, parenteral, intravenous, intranasal, subcutaneous,and intramuscular administration and formulation.

In certain applications, the pharmaceutical or therapeutic compositionsof the invention do not stimulate an immune reaction. In otherembodiments, the pharmaceutical or therapeutic compositions of theinvention, typically comprising one or more AARS polypeptides orpolynucleotides, stimulate an immune reaction, such as by serving as anadjuvant in a vaccine or related composition, or being present in acomposition together with a separate adjuvant or agent stimulates animmune response.

In certain embodiments, the AARS agents such as AARS polypeptides, AARSpolynucleotides, and antibodies have a solubility that is desirable forthe particular mode of administration, such intravenous administration.Examples of desirable solubilities include at least about 1 mg/ml, atleast about 10 mg/ml, at least about 25 mg/ml, and at least about 50mg/ml.

In certain applications, the pharmaceutical compositions disclosedherein may be delivered via oral administration to a subject. As such,these compositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain circumstances it will be desirable to deliver thepharmaceutical compositions disclosed herein parenterally,subcutaneously, intravenously, intramuscularly, intra-arterially,intrathecally, intraparenchymally, intracisternally,intraventricularlly, intraurethrally, intrasternally, intracranially,intrasynovially, or even intraperitoneally as described, for example, inU.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515 and U.S. Pat. No.5,399,363 (each specifically incorporated herein by reference in itsentirety). Suitable devices for parenteral administration include needle(including microneedle) injectors, needle-free injectors, and infusiontechniques.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form should be sterileand should be fluid to the extent that easy syringability exists. Itshould be stable under the conditions of manufacture and storage andshould be preserved against the contaminating action of microorganisms,such as bacteria and fungi. The carrier can be a solvent or dispersionmedium containing, for example, water, ethanol, polyol (e.g., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be facilitated by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. In thisconnection, a sterile aqueous medium that can be employed will be knownto those of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in 1 ml of isotonic NaCl solutionand either added to 1000 ml of hypodermoclysis fluid or injected at theproposed site of infusion (see, e.g., Remington's PharmaceuticalSciences, 15th Edition, pp. 1035-1038 and 1570-1580). Some variation indosage will necessarily occur depending on the condition of the subjectbeing treated. The person responsible for administration will, in anyevent, determine the appropriate dose for the individual subject.Moreover, for human administration, preparations should meet sterility,pyrogenicity, and the general safety and purity standards as required byFDA Office of Biologics standards.

Sterile injectable solutions can be prepared by incorporating the activecompounds in the required amount in the appropriate solvent with thevarious other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions disclosed herein may be formulated in a neutral or saltform. Pharmaceutically-acceptable salts, include the acid addition salts(formed with the free amino groups of the protein) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups can also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, histidine, procaine and the like. Upon formulation,solutions will be administered in a manner compatible with the dosageformulation and in such amount as is therapeutically effective. Theformulations are easily administered in a variety of dosage forms suchas injectable solutions, drug-release capsules, and the like.

As used herein, “carrier” includes any and all solvents, dispersionmedia, vehicles, coatings, diluents, antibacterial and antifungalagents, isotonic and absorption delaying agents, buffers, carriersolutions, suspensions, colloids, and the like. The use of such mediaand agents for pharmaceutical active substances is well known in theart. Except insofar as any conventional media or agent is incompatiblewith the active ingredient, its use in the therapeutic compositions iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

The phrase “pharmaceutically-acceptable” refers to molecular entitiesand compositions that do not produce an allergic or similar untowardreaction when administered to a human. The preparation of an aqueouscomposition that contains a protein as an active ingredient is wellunderstood in the art. Typically, such compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid prior to injectioncan also be prepared. The preparation can also be emulsified.

In certain embodiments, the pharmaceutical compositions may be deliveredby intranasal sprays, inhalation, and/or other aerosol deliveryvehicles. Methods for delivering genes, polynucleotides, and peptidecompositions directly to the lungs via nasal aerosol sprays have beendescribed e.g., in U.S. Pat. No. 5,756,353 and U.S. Pat. No. 5,804,212(each specifically incorporated herein by reference in its entirety).Likewise, the delivery of drugs using intranasal microparticle resins(Takenaga et al., 1998) and lysophosphatidyl-glycerol compounds (U.S.Pat. No. 5,725,871, specifically incorporated herein by reference in itsentirety) are also well-known in the pharmaceutical arts. Likewise,transmucosal drug delivery in the form of a polytetrafluoroetheylenesupport matrix is described in U.S. Pat. No. 5,780,045 (specificallyincorporated herein by reference in its entirety).

The pharmaceutical compositions may be formulated to be immediate and/orsustained release. Sustained release compositions include delayed,modified, pulsed, controlled, targeted and programmed release. Thus thecompositions may be formulated as a suspension or as a solid,semi-solid, or thixotropic liquid for administration as an implanteddepot providing sustained release of the AARS polynucleotides, AARSpolypeptides, binding agents, modulatory agents and other active agents.Examples of such formulations include without limitation, drug-coatedstents and semi-solids and suspensions comprising drug-loadedpoly(DL-lactic-co-glycolic)acid (PGLA), poly(DL-lactide-co-glycolide)(PLG) or poly(lactide) (PLA) lamellar vesicles or microparticles,hydrogels (Hoffman A S: Ann. N.Y. Acad. Sci. 944: 62-73 (2001)),poly-amino acid nanoparticles systems, sold under the trademark MEDUSA®developed by Flamel Technologies Inc., non aqueous gel systems soldunder the trademark ATRIGEL® developed by Atrix, Inc., and SucroseAcetate Isobutyrate Extended Release formulations sold under thetrademark SABER® developed by Durect Corporation, and lipid-basedsystems developed by SkyePharma and sold under the trademark DEPOFOAM®.

Sustained release devices capable of delivering desired doses of thepharmaceutical compositions over extended periods of time are known inthe art. For example, U.S. Pat. Nos. 5,034,229; 5,557,318; 5,110,596;5,728,396; 5,985,305; 6,113,938; 6,156,331; 6,375,978; and 6,395,292;teach osmotically-driven devices capable of delivering an active agentformulation, such as a solution or a suspension, at a desired rate overan extended period of time (i.e., a period ranging from more than oneweek up to one year or more). Other exemplary sustained release devicesinclude regulator-type pumps that provide constant flow, adjustableflow, or programmable flow of beneficial agent formulations, which areavailable from Medtronic including the Intrathecal pumps sold under thetrademark SYNCHROMED INFUSION SYSTEM®, the Johnson and Johnson systemssold under the trademark CODMAN® division pumps, and INSET® technologiespumps. Further examples of devices are described in U.S. Pat. Nos.6,283,949; 5,976,109; 5,836,935; and 5,511,355.

In certain embodiments, the delivery may occur by use of liposomes,nanocapsules, microparticles, microspheres, lipid particles, vesicles,and the like, for the introduction of the compositions of the presentinvention into suitable host cells. In particular, the compositions ofthe present invention may be formulated for delivery either encapsulatedin a lipid particle, a liposome, a vesicle, a nanosphere, a nanoparticleor the like. The formulation and use of such delivery vehicles can becarried out using known and conventional techniques.

In certain embodiments, the agents provided herein may be attached to apharmaceutically acceptable solid substrate, including biocompatible andbiodegradable substrates such as polymers and matrices. Examples of suchsolid substrates include, without limitation, polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such aspoly(lactic-co-glycolic acid) (PLGA) and the LUPRON DEPOT™ (injectablemicrospheres composed of lactic acid-glycolic acid copolymer andleuprolide acetate), poly-D-(−)-3-hydroxybutyric acid, collagen, metal,hydroxyapatite, bioglass, aluminate, bioceramic materials, and purifiedproteins.

In one particular embodiment, the solid substrate comprisesbiodegradable polymers sold under the trademark ATRIGEL™ (QLT, Inc.,Vancouver, B.C.). The ATRIGEL® drug delivery system consists ofbiodegradable polymers dissolved in biocompatible carriers.Pharmaceuticals may be blended into this liquid delivery system at thetime of manufacturing or, depending upon the product, may be added laterby the physician at the time of use. When the liquid product is injectedinto the subcutaneous space through a small gauge needle or placed intoaccessible tissue sites through a cannula, water in the tissue fluidscauses the polymer to precipitate and trap the drug in a solid implant.The drug encapsulated within the implant is then released in acontrolled manner as the polymer matrix biodegrades with time.

Pharmaceutical compositions for use in the present invention may also beadministered topically, (intra)dermally, or transdermally to the skin ormucosa. Typical formulations for this purpose include gels, hydrogels,lotions, solutions, creams, ointments, dusting powders, dressings,foams, films, skin patches, wafers, implants, sponges, fibers, bandages,and microemulsions. Liposomes may also be used. Typical carriers includealcohol, water, mineral oil, liquid petrolatum, white petrolatum,glycerin, polyethylene glycol, and propylene glycol. Penetrationenhancers may be incorporated—see, e.g., Finnin and Morgan: J. Pharm.Sci. 88(10): 955-958, (1999). Other means of topical administrationinclude delivery by electroporation, iontophoresis, phonophoresis,sonophoresis, and microneedle or needle-free injection for example usingthe systems sold under the trademarks POWDERJECT™, and BIOJECT™.

Methods of formulation are well known in the art and are disclosed, forexample, in Remington: The Science and Practice of Pharmacy, MackPublishing Company, Easton, Pa., 20th edition, ISBN: 0683306472 (2000).The compositions and agents provided herein may be administeredaccording to the methods of the present invention in any therapeuticallyeffective dosing regimen. The dosage amount and frequency are selectedto create an effective level of the agent without harmful effects. Theeffective amount of a compound of the present invention will depend onthe route of administration, the type of warm-blooded animal beingtreated, and the physical characteristics of the specific warm-bloodedanimal under consideration. These factors and their relationship todetermining this amount are well known to skilled practitioners in themedical arts. This amount and the method of administration can betailored to achieve optimal efficacy but will depend on such factors asweight, diet, concurrent medication and other factors which thoseskilled in the medical arts will recognize.

In particular embodiments, the amount of a composition or agentadministered will generally range from a dosage of from about 0.1 toabout 100 mg/kg/day, and typically from about 0.1 to 10 mg/kg whereadministered orally or intravenously. In particular embodiments, adosage is 5 mg/kg or 7.5 mg/kg. In various embodiments, the dosage isabout 50-2500 mg per day, 100-2500 mg/day, 300-1800 mg/day, or 500-1800mg/day. In one embodiment, the dosage is between about 100 to 600mg/day. In another embodiment, the dosage is between about 300 and 1200mg/day. In particular embodiments, the composition or agent isadministered at a dosage of 100 mg/day, 240 mg/day 300 mg/day, 600mg/day, 1000 mg/day, 1200 mg/day, or 1800 mg/day, in one or more dosesper day (i.e., where the combined doses achieve the desired dailydosage). In related embodiments, a dosage is 100 mg bid, 150 mg bid, 240mg bid, 300 mg bid, 500 mg bid, or 600 mg bid. In various embodiments,the composition or agent is administered in single or repeat dosing. Theinitial dosage and subsequent dosages may be the same or different.

In certain embodiments, a composition or agent is administered in asingle dosage of 0.1 to 10 mg/kg or 0.5 to 5 mg/kg. In otherembodiments, a composition or agent is administered in a dosage of 0.1to 50 mg/kg/day, 0.5 to 20 mg/kg/day, or 5 to 20 mg/kg/day.

In certain embodiments, a composition or agent is administered orally orintravenously, e.g., by infusion over a period of time of about, e.g.,10 minutes to 90 minutes. In other related embodiments, a composition oragent is administered by continuous infusion, e.g., at a dosage ofbetween about 0.1 to about 10 mg/kg/hr over a time period. While thetime period can vary, in certain embodiments the time period may bebetween about 10 minutes to about 24 hours or between about 10 minutesto about three days.

In particular embodiments, an effective amount or therapeuticallyeffective amount is an amount sufficient to achieve a totalconcentration of the composition or agent in the blood plasma of asubject with a C_(max) of between about 0.1 μg/ml and about 20 μg/ml orbetween about 0.3 μg/ml and about 20 μg/ml. In certain embodiments, anoral dosage is an amount sufficient to achieve a blood plasmaconcentration (C_(max)) of between about 0.1 μg/ml to about 5 μg/ml orbetween about 0.3 pg/ml to about 3 μg/ml. In certain embodiments, anintravenous dosage is an amount sufficient to achieve a blood plasmaconcentration (C_(max)) of between about 1 μg/ml to about 10 μg/ml orbetween about 2 μg/ml and about 6 μg/ml. In a related embodiment, thetotal concentration of an agent in the blood plasma of the subject has amean trough concentration of less than about 20 μg/ml and/or a steadystate concentration of less than about 20 μg/ml. In a furtherembodiment, the total concentration of an agent in the blood plasma ofthe subject has a mean trough concentration of less than about 10 μg/mland/or a steady state concentration of less than about 10 μg/ml.

In yet another embodiment, the total concentration of an agent in theblood plasma of the subject has a mean trough concentration of betweenabout 1 ng/ml and about 10 μg/ml and/or a steady state concentration ofbetween about 1 ng/ml and about 10 μg/ml. In one embodiment, the totalconcentration of an agent in the blood plasma of the subject has a meantrough concentration of between about 0.3 μg/ml and about 3 μg/ml and/ora steady state concentration of between about 0.3 μg/ml and about 3μg/ml.

In particular embodiments, a composition or agent is administered in anamount sufficient to achieve in the mammal a blood plasma concentrationhaving a mean trough concentration of between about 1 ng/ml and about 10μg/ml and/or a steady state concentration of between about 1 ng/ml andabout 10 μg/ml. In related embodiments, the total concentration of theagent in the blood plasma of the mammal has a mean trough concentrationof between about 0.3 μg/ml and about 3 μg/ml and/or a steady stateconcentration of between about 0.3 μg/ml and about 3 μg/ml.

In particular embodiments of the present invention, the effective amountof a composition or agent, or the blood plasma concentration ofcomposition or agent is achieved or maintained, e.g., for at least 15minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes,at least 90 minutes, at least 2 hours, at least 3 hours, at least 4hours, at least 8 hours, at least 12 hours, at least 24 hours, at least48 hours, at least 3 days, at least 4 days, at least 5 days, at least 6days, at least one week, at least 2 weeks, at least one month, at least2 months, at least 4 months, at least 6 months, at least one year, atleast 2 years, or greater than 2 years.

In certain polypeptide-based embodiments, the amount of polypeptideadministered will typically be in the range of about 0.1 μg/kg to about0.1 mg/kg to about 50 mg/kg of patient body weight. Depending on thetype and severity of the disease, about 0.1 μg/kg to about 0.1 mg/kg toabout 50 mg/kg body weight (e.g., about 0.1-15 mg/kg/dose) ofpolypeptide can be an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. For example, a dosing regimen may compriseadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of the polypeptide, or abouthalf of the loading dose. However, other dosage regimens may be useful.A typical daily dosage might range from about 0.1 μg/kg to about 1 μg/kgto 100 mg/kg or more, depending on the factors mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofdisease symptoms occurs.

In particular embodiments, the effective dosage achieves the bloodplasma levels or mean trough concentration of a composition or agentdescribed herein. These may be readily determined using routineprocedures.

Embodiments of the present invention, in other aspects, provide kitscomprising one or more containers filled with one or more of thepolypeptides, polynucleotides, antibodies, multiunit complexes,compositions thereof, etc., of the invention, as described herein. Thekits can include written instructions on how to use such compositions(e.g., to modulate cellular signaling, angiogenesis, cancer,inflammatory conditions, diagnosis etc.).

The kits herein may also include a one or more additional therapeuticagents or other components suitable or desired for the indication beingtreated, or for the desired diagnostic application. An additionaltherapeutic agent may be contained in a second container, if desired.Examples of additional therapeutic agents include, but are not limitedto anti-neoplastic agents, anti-inflammatory agents, antibacterialagents, antiviral agents, angiogenic agents, etc.

The kits herein can also include one or more syringes or othercomponents necessary or desired to facilitate an intended mode ofdelivery (e.g., stents, implantable depots, etc.).

All publications, patent applications, and issued patents cited in thisspecification are herein incorporated by reference as if each individualpublication, patent application, or issued patent were specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims. The following examples are provided byway of illustration only and not by way of limitation. Those of skill inthe art will readily recognize a variety of noncritical parameters thatcould be changed or modified to yield essentially similar results.

XIV. EXAMPLES General Methods

Unless indicated otherwise in the examples below, the following generalmethods for gene optimization, small and large scale protein expression,protein purification, transcriptional profiling and screening were usedto make and characterize the AARS polypeptides described in the Examplesbelow.

Gene Synthesis and Cloning into Expression Vectors

Polynucleotide sequences encoding epitope tagged versions of the AARSpolypeptides were codon optimized and cloned into bacterial expressionvectors using the methods listed below.

In method (1), E. coli codon-optimized DNA (Welch et al., PLoS ONE 4(9):e7007 doi:10.1371/journal.pone.0007002) encoding each AARS polypeptideis synthesized by DNA 2.0 (Menlo Park, Calif.), and two versions of eachAARS polypeptide are synthesized, containing either an N-terminal, orC-terminal combined epitope tag comprising both a six histidine tag andV5 epitope tag.

DNA encoding the N-terminally tagged AARS polypeptides is synthesizedwith a 5′ extension encoding in 5′ to 3′ orientation, a ribosome bindingsite (rbs (underlined below)), NdeI restriction site, six histidine tag,and a V5 epitope tag,

(SEQ. ID. No. 1) (AGGAGGTAAAACATATGCATCATCATCATCATCACGGTAAGCCTATCCCTAACCCTTTGCTCGGTCTCGATTCTACG),

which is fused in frame to the predicted AARS polypeptide open readingframe. In cases where the AARS polypeptide comprises a predicted nativeinitiation methionine (ATG) residue, or the first amino acid residue ofthe predicted AARS polypeptide is Met, this was deleted. At the end ofthe predicted AARS polypeptide open reading frame, two stop codons and aXhoI site (TAATGACTCGAG) (SEQ. ID. No. 2) are added.

DNA encoding the C-terminally tagged AARS polypeptides is synthesizedwith a 5′ extension encoding a rbs (underlined below) and NdeIrestriction site that either recapitulates the predicted native startcodon for the AARS polypeptide, or inserts an ATG in frame with thepredicted AARS polypeptide open reading frame,

(SEQ. ID. No. 3) (AGGAGATAAAACATATG)

In different embodiments, the ribosome binding site can comprise thesequences “AGGAGGTAAAACAT” (SEQ. ID. No. 4), “AGGAGATAAAACAT” (SEQ. ID.No. 5), or GAAGGAGATATACAT (SEQ. ID. No. 6). At the 3′ end of thepredicted AARS polypeptide open reading frame, a 3′ extension issynthesized which encodes in 5′ to 3′ order, a V5 epitope tag, sixhistidine tag, two stop codons and a XhoI site,(GGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCACCACCATCATCACCATTAATGACTCGAG) (SEQ. ID. No. 7), which is fused in frame to thepredicted AARS polypeptide open reading frame. If the AARS polypeptideincluded a predicted native stop codon, this was deleted.

Synthesized DNA sequences encoding the AARS polypeptides are subclonedinto pJExpress411 vector (DNA 2.0). After sequencing to confirmsynthesis of the correct product, expression vectors are transformedinto bacteria for protein expression as described more fully below.

In method (2), E. coli codon-optimized DNA (Ermolaeva Md. (2001) Curr.Iss. Mol. Biol. 3 (4) 91-7) encoding each AARS polypeptide issynthesized by GENEWIZ (South Plainfield, N.J.). Each polynucleotidesequence encoding the AARS polypeptide was synthesized with short 5′ and3′ extensions comprising unique restriction sites for subsequentcloning.

Specifically a BamHI restriction site was inserted at the 5′ end of thepredicted open reading frame. In cases where the AARS polypeptidecomprises a predicted native initiation methionine residue (ATG), or thefirst amino acid residue of the predicted AARS polypeptide is Met, thiswas deleted. Additionally a XhoI restriction site was inserted at the 3′end of the predicted open reading frame. In cases where the AARSpolypeptide comprises a predicted native stop codon, this was deleted.

After restriction digestion, the resulting DNA sequences are subclonedinto modified pET-24b vectors (EMD, Gibbstown, N.J.) containing eitheran N-terminal (pET24b_N-6×HisN5), or C-terminal (pET24b_C-V5/6×His)combined epitope tag comprising both a six histidine and V5 epitope tag(vector modification by GENEWIZ, (South Plainfield, N.J.).

After restriction digestion, and cloning, the DNA encoding the N-taggedAARS polypeptide is cloned into the N-tagged vector (pET24b_N-6×His/V5),which comprises a 5′ DNA sequence encoding six histidines and a V5epitope tag,(CATATGCATCATCATCATCATCACGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGGGATCC) (SEQ. ID. No. 8), in frame with an initiation codon (ATG)embedded within the NdeI restriction site. This 5′ extension is fused tothe predicted AARS polypeptide open reading frame through a short 2amino acid linker (GS).

At the 3′ end of the predicted open reading frame, the DNA encoding theN-tagged AARS polypeptide comprises a DNA sequence encoding a 2 aminoacid extension (LE) followed by two termination codons (CTCGAGTAATGA)(SEQ. ID. No. 9).

After restriction digestion, and cloning, the DNA encoding the C-taggedAARS polypeptide cloned into the C-tagged vector (pET24b_C-V5/6×His),comprises a 5′ sequence encoding an initiation codon (ATG) embeddedwithin the NdeI restriction site which is fused to the predicted AARSpolypeptide open reading frame through a short 2 amino acid linker (GS),(CATATGGGATCC) (SEQ. ID. No. 10).

At the 3′ end of the predicted open reading frame, the DNA encoding theC-tagged AARS polypeptide comprises a 3′ DNA sequence encoding a shortlinker 2 amino acid linker (LE) followed by a V5 epitope tag followed bysix histidines, and two stop codons,CTCGAGGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGCACCACCACCACCACCACTAATGA (SEQ. ID. No. 11).

AARS Polypeptide Expression, Purification and BiophysicalCharacterization

6×His-tagged AARS polypeptides are expressed in bacteria in amedium-throughput format and/or in larger scale flask cultures dependingupon the amount of protein required. AARS polypeptides are purifiedusing affinity and ion exchange chromatography as described below, andas specified for specific experiments.

Bacterial Cultures:

100 ng of expression vector comprising codon optimized DNA encoding eachAARS polypeptide (as described above) is transformed into BL21(DE3) (EMDchemicals, cat. no. 69450) competent E. coli bacteria at 42° C. for 30seconds in PCR plates. C41(DE3) (Lucigen, cat. no. 60442), HMS174(DE3)(EMD chemicals, cat. no. 69453) and Origami2(DE3) (EMD chemicals, cat.no. 71345) strains are also evaluated. The plates are placed on ice for2 minutes and 100 μL of SOC medium is added, followed by a 1-hourincubation at 37° C. 5 mL of auto-induction medium (EMD chemicals, cat.no. 71491) supplemented with kanamycin (100 μg/mL) is added into eachwell of a 24-well block (Qiagen, cat. no. 19583). The transformationreactions are added to the individual wells, the block is sealed withadhesive film (VWR, cat. no 60941-078) and incubated overnight at 250rpm in a 37° C. shaker. When low temperature (25° C.) conditions areused, incubation is carried out for 48 hours instead.

For larger scale expression, 200 mL of auto-induction mediumsupplemented with kanamycin (100 μg/mL) is added into 500-mL Erlenmeyerflasks with vent caps (Corning, cat. no. 431401). The transformationreactions are added to the individual flasks and incubated for 30 hoursat 250 rpm in a 37° C. shaker.

Protein Isolation:

After the culture reached stationary phase (typical OD₆₀₀ of 3-6), theblocks are centrifuged at 3600×g for 10 minutes. The medium is carefullyaspirated and the blocks are frozen at −80° C. or −20° C. for 10minutes. The blocks are then allowed to thaw at room temperature and 1mL lysis buffer (100 mL Bugbuster supplemented with 200 μL, lysonase(EMD chemicals, cat. no 71370) and protease inhibitors “complete miniEDTA-free” (Roche, cat. no. 11 836 170 001)) is added into each well.The pellets are resuspended by repeat pipetting until no clump isvisible and transferred to eppendorf tubes, followed by a 10-20 minuteincubation on a shaker at room temperature. After centrifugation at16,000 g for 10 minutes at 4° C., the lysates are loaded onto aTurboFilter 96 Plate included in the Ni-NTA Superflow 96 BioRobot Kit(Qiagen, cat. no. 969261) and centrifuged at 500 g for 5-10 minutes.

For larger scale expression, the stationary phase culture is transferredinto 500-mL bottles and centrifuged at 6,000 g for 10 minutes. Themedium is decanted and the pellet is stored at −80° C. or −20° C. beforefurther processing. The pellet is then allowed to thaw at roomtemperature and 20 mL lysis buffer is added into each bottle. Thepellets are resuspended by repeat pipetting until no clump is visible,followed by 20 minute incubation on a shaker at room temperature. Aftercentrifugation at 10,000 g for 30 minutes at 4° C., the lysates aretransferred to clean tubes or bottles. If trace amounts of debris arecarried over during the transfer, the sample is centrifuged again orpassed through a 0.45 μm cellulose acetate membrane (Corning, cat. no.430314) for further clarification.

Affinity Purification:

A QIAFilter 96 Plate is loaded with 200 μL Ni-NTA Superflow slurryincluded in the Ni-NTA Superflow 96 BioRobot Kit and the resin isequilibrated by adding 600 μL binding buffer (20 mM sodium phosphate,500 mM sodium chloride and 10 mM imidazole, pH 7.5). A vacuum of −15 in.Hg is applied until all the buffer has passed through the resin. Theclarified cell lysates from the previous step are then loaded onto theQIAFilter® 96 Plate and allowed to bind for 5 minutes. A vacuum of −3in. Hg is applied for approximately 5 minutes until all the samples havepassed through the resin. The resin is then washed with 1 mL bindingbuffer, followed by two washes with 1 mL binding buffer containing 0.1%Triton X-100. The resin is then washed 10 times with 1 mL binding bufferwithout Triton X-100. The bound 6×His-tagged AARS polypeptides areeluted with 450 μL elution buffer (20 mM sodium phosphate, 500 mM sodiumchloride and 500 mM imidazole, pH 7.5) and stored at 4° C.

For larger scale expression, an empty disposable column “Poly-Prep”(Bio-Rad, cat. no. 731-1550) is loaded with 1 mL Ni-NTA Superflow slurry(Qiagen, cat. no. 30450) and the 0.5 mL resin is equilibrated by adding5 mL binding buffer. The clarified cell lysate from the previous step isthen loaded onto the column and allowed to pass through by gravity. Theresin is first washed with 50 mL binding buffer plus 0.1% Triton X-100,then washed with 50 mL binding buffer without Triton X-100. The bound6×His-tagged AARS polypeptides are eluted with 2 mL elution buffer andstored at 4° C.

Desalting and Polishing Steps:

For AARS polypeptides with a molecular mass of >10 kDa, the Omega 10Kmembrane of an AcroPrep 96 filter plate (Pall, cat. no. 5034) is rinsedwith 20 μL 1×PBS and the plate is placed onto a vacuum manifold (>10 inHg) until all the liquid passes through. The eluates from the previousstep (Ni-NTA) are dispensed into each well and the vacuum applied untilall the liquid passes through. These steps are repeated until the totaleluate volume (450 μL) has been processed. AARS polypeptides arerecovered by adding 180 μL of 1×PBS pH 7.4 to each well, pipetting upand down 10 times carefully and then transferred to a clean block. Thisstep is repeated to yield a total volume of 360 μL per well and theblock is stored at 4° C. For AARS polypeptides with a molecular mass of<10 kDa, the eluates from Ni-NTA are loaded onto an Amicon Ultra-15Centrifugal Filter Unit with Ultracel-3 membrane (Millipore, cat. no.UFC900308), followed by the addition of 10 mL 1×PBS and a centrifugationat 3,600 g for 10-30 minutes until the volume is less than 360 μL. Thesamples are recovered and 1×PBS is added to a final volume of 360 μL.

In order to remove endotoxins, an AcroPrep Advance filter plate withMustang Q membrane (Pall, cat. no. 8171) is rinsed with 300 μL of 1×PBSand centrifuged at 1,000 g for 5 minutes to remove the buffer. Thedesalted AARS polypeptides (360 μL/well) are added to the filter plateand incubated on a shaker for 5-10 minutes. The plate is thencentrifuged at 1,000 g for 5-10 minutes and the flow through fractionscontaining the AARS polypeptides are collected and stored at 4° C.

For larger scale expression, the eluates from Ni-NTA are loaded onto anAmicon Ultra-15 Centrifugal Filter Unit with Ultracel-3 or Ultracel-10membrane (Millipore, cat. no. UFC900308 or UFC901008) depending on themolecular weight of the AARS polypeptide and then centrifuged at 3,600 gfor 10-30 minutes until the volume is reduced to 250 μL. The samples aremixed in 10 mL 1×PBS, pH7.4 and centrifuged again at 3,600 g for 10-30minutes until the volume is about 250 μL. This step is repeated one moretime, the supernatants are recovered and 1×PBS is added to a finalvolume of 1.5 mL.

In order to remove endotoxins, a Sartobind Q 5 strong anion exchangermembrane (Sartorius, cat. no. Q5F) is flushed with 1 mL 1×PBS and theAARS polypeptides are slowly passed through the membrane using a plasticsyringe. The flow through fraction containing the AARS polypeptides iscollected in a 96-deep well block that is sealed and stored at 4° C.

6×His-tagged AARS polypeptides expressed in bacteria and found ininclusion bodies are purified using affinity chromatography and a seriesof refolding steps, as described below.

Bacterial Cultures:

100 ng of plasmid encoding each AARS polypeptide is transformed intoBL21(DE3) (EMD chemicals, cat. no. 69450) or C41(DE3) (Lucigen, cat. no.60442) competent E. coli bacteria at 42° C. for 30 seconds in PCRplates. The plates are placed on ice for 2 minutes and 100 μL, of SOCmedium is added, followed by a 1-hour incubation at 37° C. 5 mL ofauto-induction medium (EMD chemicals, cat. no. 71491) supplemented withkanamycin (100 μg/mL) is added into each well of a 24-well block(Qiagen, cat. no. 19583). The transformation reactions are added to theindividual wells, the block is sealed with adhesive film (VWR, cat. no60941-078) and incubated overnight at 250 rpm in a 37° C. shaker.

For larger scale expression, 200 mL of auto-induction mediumsupplemented with kanamycin (100 μg/mL) is added into 500-mL Erlenmeyerflasks with vent caps (Corning, cat. no. 431401). The transformationreactions are added to the individual flasks and incubated for 30 hoursat 250 rpm in a 37° C. shaker.

Isolation:

After the cultures reach stationary phase (typical OD₆₀₀ of 3-6), theblocks are centrifuged at 3,600×g for 10 minutes. The medium iscarefully aspirated and the blocks are frozen at −80° C. or −20° C. for10 minutes. The blocks are then allowed to thaw at room temperature and1 mL lysis buffer (100 mL Bugbuster supplemented with 200 μl lysonase(EMD chemicals, cat. no 71370) and protease inhibitor “complete miniEDTA-free” (Roche, cat. no. 11 836 170 001)) is added into each well.The pellets are resuspended by repeat pipetting until no clump isvisible and transferred to eppendorf tubes, followed by a 10-20 minuteincubation on a shaker at room temperature. After centrifugation at16,000×g for 10 minutes at 4° C., the soluble lysates are discarded andthe inclusion bodies are thoroughly resuspended in denaturing bindingbuffer (20 mM sodium phosphate, 500 mM sodium chloride, 6 M guanidinehydrochloride, 10 mM imidazole, pH 7.5). The samples are centrifuged at16,000 g for 10 minutes and the supernatants loaded onto a TurboFilter96 Plate included in the Ni-NTA Superflow 96 BioRobot Kit (Qiagen, cat.no. 969261) followed by centrifugation at 500 g for 5-10 minutes. Thefiltrates are collected in a clean 96-well block (Greiner, cat. no.780286).

For larger scale expression, the stationary phase culture is transferredinto 500-mL bottles and centrifuged at 6,000 g for 10 minutes. Themedium is decanted and the pellet is stored at −80° C. or −20° C. beforefurther processing. The pellet is then allowed to thaw at roomtemperature and 20 mL lysis buffer is added into each bottle. Thepellets are resuspended by repeat pipetting until no clump is visible,followed by 20 minute incubation on a shaker at room temperature. Aftercentrifugation at 10,000 g for 30 minutes at 4° C., the soluble lysatesare discarded and the insoluble inclusion bodies thoroughly resuspendedin denaturing binding buffer.

Affinity Purification: A QIAFilter 96 Plate is loaded with 200 μL Ni-NTASuperflow slurry included in the Ni-NTA Superflow 96 BioRobot Kit andthe resin is equilibrated by adding 600 μL denaturing binding buffer(see above). A vacuum of −15 in. Hg is applied until all of the bufferpasses through the resin. The clarified denatured samples from theprevious step are then loaded onto the QIAFilter® 96 Plate and allowedto bind for 5 minutes. A vacuum of approximately 3 inches of mercury isapplied for approximately 5 minutes until all the samples pass throughthe resin. The resin is then washed with 1 mL denaturing binding buffer,followed by five washes with 1 mL denaturing binding buffer containing0.1% Triton X-100. The resin is then washed 15 times with 1 mLdenaturing binding buffer without Triton X-100. The bound 6×His-taggedAARS polypeptides are then eluted with 450 μL denaturing elution buffer(20 mM sodium phosphate, 500 mM sodium chloride, 6 M guanidinehydrochloride and 500 mM imidazole, pH 7.5) and stored at 4° C.

For larger scale expression, an empty disposable column “Poly-Prep”(Bio-Rad, cat. no. 731-1550) is loaded with 1 mL Ni-NTA Superflow slurry(Qiagen, cat. no. 30450) and the 0.5 mL resin is equilibrated by adding5 mL denaturing binding buffer (see above). The denatured inclusionbodies from the previous step are then loaded onto the column andallowed to pass through by gravity. The resin is first washed with 50 mLdenaturing binding buffer plus 0.1% Triton X-100, then washed with 50 mLdenaturing binding buffer without Triton X-100. The bound 6×His-taggedAARS polypeptides are eluted with 2 mL denaturing elution buffer andstored at 4° C.

Refolding:

For AARS polypeptides >10 kDa, the Omega 10K membrane of an AcroPrep 96filter plate (Pall, cat. no. 5034) is rinsed with 20 μL, 1×PBS and theplate is placed onto a vacuum manifold (>10 in. Hg) until all the liquidpasses through. The eluates from the previous step (Ni-NTA) aredispensed into each well and the vacuum applied until all the liquidpasses through. These steps are repeated until the total eluate volume(450 μL) has been processed. AARS polypeptides are recovered by adding200 μL, of refolding buffer containing 50 mM Tris, 250 mM sodiumchloride, 10 mM potassium chloride, 2 mM magnesium chloride, 2 mMcalcium chloride, 400 mM sucrose, 500 mM arginine, 1 mM DTT and 0.01%polysorbate 80, pH 7.4) to each well, pipetting up and down 10 timescarefully, and then transferred to a clean block. This step is repeatedto yield a total volume of 400 μL, per well and the block is placed onthe shaker overnight at 4° C. For AARS polypeptides <10 kDa, the eluatesfrom Ni-NTA are loaded onto an Amicon Ultra-15 Centrifugal Filter Unitwith Ultracel-3 membrane (Millipore, cat. no. UFC900308), followed bythe addition of 10 mL refolding buffer and a centrifugation at 3,600 gfor 10-30 minutes until the volume is less than 400 μL. The samples arerecovered and extra refolding buffer is added to a final volume of 4004. The samples are transferred to a 96-well block, sealed with film andplaced on a shaker overnight at 4° C.

For larger scale cultures, the eluates from Ni-NTA are loaded onto anAmicon Ultra-15 centrifugal filter unit with Ultracel-3 or Ultracel-10membrane (Millipore, cat. no. UFC900308 or UFC901008 depending on themolecular weight of the AARS polypeptide) and then centrifuged at 3,600g for 10-30 minutes until the volume is reduced to about 500 μL. ForAARS polypeptides with pI>7, the samples are diluted 20-fold in thefollowing buffer: 50 mM sodium acetate, 10 mM sodium chloride, 0.4 mMpotassium chloride, 1 mM EDTA, 400 mM sucrose, 500 mM arginine, 1 mM DTTand 0.01% polysorbate 80, pH 6.0. For AARS polypeptides with pI<7, thesamples are diluted 20-fold in the following buffer: 50 mM Tris, 250 mMsodium chloride, 10 mM potassium chloride, 2 mM magnesium chloride, 2 mMcalcium chloride, 400 mM sucrose, 500 mM arginine, 1 mM DTT and 0.01%polysorbate 80, pH 8.0. The samples are incubated on a shaker at 4° C.overnight.

Desalting and Polishing Steps:

After overnight incubation, the 96-well block is centrifuged at 3,600 gto remove any potential aggregates. The supernatants are then subjectedto buffer exchange with 1×PBS (Invitrogen, cat. no. 10010). For AARSpolypeptides >10 kDa, the Omega 10K membrane of an AcroPrep 96 filterplate is rinsed with 20 μL 1×PBS and the plate is placed onto a vacuummanifold (>10 in. Hg) until all the liquid passes through. The samplesin the refolding buffer are dispensed into each well and the vacuumapplied until all the liquid passes through. These steps are repeateduntil the total sample volume (400 μL) has been processed. AARSpolypeptides are recovered by adding 180 μL of 1×PBS pH 7.4 to eachwell, pipetting up and down 10 times carefully, and then transferred toa clean block. This step is repeated to yield a total volume of 360 μLper well and the block is stored at 4° C. For AARS polypeptides <10 kDa,the refolded samples are loaded onto an Amicon Ultra-15 CentrifugalFilter Unit with Ultracel-3 membrane (Millipore, cat. no. UFC900308)followed by the addition of 10 mL 1×PBS and centrifugation at 3,600 gfor 10-30 minutes until the volume is less than 360 μL. The samples arerecovered and 1×PBS is added to a final volume of 360 μL.

In order to remove endotoxins, an AcroPrep Advance filter plate withMustang Q membrane (Pall, cat. no. 8171) is rinsed with 300 μL of 1×PBSand centrifuged at 1,000 g for 5 minutes to remove the buffer. The AARSpolypeptides (360 μL/well) are added to the filter plate and incubatedon a shaker for 5-10 minutes. The plate is then centrifuged at 1,000 gfor 5-10 minutes and the flow through fractions containing the AARSpolypeptides are collected and stored at 4° C.

For larger scale cultures, after overnight incubation, the refoldedsamples are centrifuged at 10,000 g for 10 minutes to remove anyinsoluble aggregates. The supernatant is loaded onto an Amicon Ultra-15Centrifugal Filter Unit and centrifuged at 3,600 g until the volume isreduced to 250 μL. The samples are mixed in 10 mL 1×PBS and centrifugedagain at 3,600 g for 10-30 minutes until the volume is about 250 μL.Note that the pH of 1×PBS is adjusted to match the pH of the refoldingbuffer, either pH 6.0 or pH 8.0. This step is repeated one more time,the supernatants are recovered and 1×PBS is added to a final volume of1.5 mL.

In order to remove endotoxins, a Sartobind Q 5 strong anion exchangermembrane (Sartorius, cat. no. Q5F) is flushed with 1 mL 1×PBS and theAARS polypeptides are slowly passed through the membrane using a plasticsyringe. The flow through fraction containing the AARS polypeptides iscollected in a 96-deep well block that is sealed and stored at 4° C.

Biophysical Characterization:

All purified AARS polypeptides are analyzed by SDS-PAGE, theirconcentration determined based on A₂₈₀ and calculated extinctioncoefficient (ProtParam on ExPASy server). Endotoxin levels are measuredby the QCL-1000 Endpoint Chromogenic LAL assay (Lonza, cat. no. 50-648U)according to the manufacturer's instructions.

Dynamic Light Scattering:

A Wyatt Technology DynaPro 99 instrument and the temperature controller(20° C.) are warmed up for 15 minutes before the experiment followed byconnection of the Dynamics software to the instrument. The acquisitiontime is set to 10 seconds for multiple acquisitions and the laser poweris set to 100%. The quartz cuvette is washed thoroughly with deionizedwater and methanol before the addition of the protein sample (15 μL at aconcentration of approximately 1 mg/mL in PBS). Air bubbles are removedby tapping the cuvette before it is inserted into the holder with thefrosted side to the left. If the intensity is too high (warning messageshown on the screen), the sample is further diluted with PBS until theintensity is decreased to a normal range. The data collected includehydrodynamic radius, polydispersity, predicted average molecular weight,percentage of intensity and percentage of mass.

Size Exclusion Chromatography:

The protein sample is diluted to a concentration of about 5-10 mg/mL inPBS before being loaded into a 100 μL sample loop on the GeneralElectric AKTA FPLC. The Superdex 200 10/300 GL size exclusion column(General Electric, cat. no. 17-5175-01) is used for separation. Thecolumn is first equilibrated with 1.5 column volume (CV) of 1×PBSbuffer, followed by sample injection. The column is run in 1 CV of 1×PBSbuffer (isocratic flow) with absorbance at 280 nm monitoring. The peakarea is integrated and the percentage calculated with the Unicornsoftware. The elution volume is used to estimate the molecular weightbased on comparison with gel filtration calibration kits (GeneralElectric, cat. no. 28-4038-41 and 28-4038-42).

Protein Recovery Upon Storage at High Concentration:

10 μL of the AARS polypeptides concentrated to ≧10 mg/mL using an AmiconUltra-15 filter unit (Millipore, cat. no. UFC901024 or UFC900324depending on molecular weight) are transferred to a cleanmicrocentrifuge tube. The sample is stored at room temperature for oneweek followed by centrifugation at 16,000 g for 10 minutes to pellet anyprecipitates. The concentration of the supernatant is determined by aBradford protein assay and compared to the concentration measured priorto the week-long exposure to room temperature. The recovery is expressedas percentage of the starting concentration.

Characterization of AARS Polypeptides by LC-MS:

Purified AARS polypeptides (1 mg/mL) are diluted 1:10 into 0.1% formicacid and 0.6 μg protein is loaded with a Dionex autosampler onto a C4capillary column. The capillary column is prepared by cutting 150 mm offused silica tubing (0.36 mm OD by 0.1 mm ID, Polymicro Technologies,cat. no. 2000023). The capillary is pulled at one end with a SuterInstrument Laser Fiber Puller and cut with a fused silica cutter togenerate a 5 μm tip. The capillary is packed to the length of 75 mm withC4 resin (5 μm, 300 Å, Michrom, cat. no. PM5/64300/00) using pressurebomb. The LC-MS analysis is performed on an ThermoFisher LTQ ion trapmass spectrometer coupled to a Dionex Ultimate3000 HPLC system. Theanalyte is eluted from the column using a 35-minute gradient of 5-70%acetonitrile in 0.1% formic acid at a flow rate of 0.9 μL/min. The LTQis operated on a full MS scan mode (300-2,000 m/z) with a spray voltageof 2.5 kV.

Data collection and analysis: raw mass spectrometry data are stored inRAW files generated by XCalibur running on the LTQ XL mass spectrometer.The MS spectra of the major peaks on the chromatograph are furtheranalyzed with ThermoFisher deconvoluting algorithm ProMass to obtain theAARS polypeptide molecular weights.

Functional Analysis of AARS Polypeptides

Transcriptional Profiling

Background and Therapeutic Relevance:

In addition to traditional target identification techniques, genomictools have recently emerged as important approaches to aid inelucidating the mechanism of action of AARS polypeptides and can providedirect insight into therapeutic relevance early in the drug discoveryprocess. To facilitate an understanding of potential therapeuticutility, primary human cell types are cultured with AARS polypeptidesand transcriptional profiling is assessed at two separate time pointsfollowing incubation with AARS polypeptides.

The cell types chosen for transcriptional profiling are based on thepluripotent capabilities of the cells in question and potential toidentify AARS polypeptides of direct therapeutic value. For example,Mesenchymal stem cells (MSCs) can differentiate into osteogenic,adipogenic, chondrogenic, myocardial, or neural lineages when exposed tospecific stimuli, making them attractive for understanding the potentialrelevance of the AARS polypeptides to a broad range of cell types, anddiseases.

In addition to supporting hematopoietic cells, marrow stromal cells canalso be induced to differentiate into cells of different connectivetissue lineage, such as bone, cartilage, and fat. The potential of HumanMesenchymal stem cells (hMSCs) to maintain multipotency and proliferateextensively in vitro provides new avenues for cell-based therapy in therestoration of damaged or diseased tissue. Recent reports also indicatethat HMSCs are capable of cell fate crossing germ layer boundaries. Inaddition to differentiating into multi-lineages of the mesoderm, thesecells can also differentiate into neurons of ectodermal origin andhepatocyte-like cells of endodermal origin. During the process ofdifferentiation, these cells may modify expression patterns of certainlineage specific transcripts.

Accordingly the ability of specific AARS polypeptides to modulatespecific patterns of genes in HMSCs in a time dependent mannerdemonstrates that these proteins play potentially significant roles in abroad array of differentiation pathways, as well as diseases anddisorders resulting from the dysfunction, or deterioration of theseprocesses, or the corresponding cell types. Moreover AARS polypeptideswith the ability to modulate gene transcription in MSCs have significanttherapeutic utility to enable the in vitro or in vivo modulation ofhematopoiesis, neurogenesis, myogenesis, osteogenesis, and adipogenesis,as well as in a broad range of disorders and diseases, including forexample inflammatory responses, autoimmunity, cancer, neuronaldegeneration, muscular dystrophy, osteoporosis, and lipodystrophy.

Human Skeletal Muscle Cells (HSkMC) can undergo differentiation toexhibit actin and myosin myofilaments, and have been used in the studyof genetic muscular diseases such as Malignant Hyperthermial. HSkMC alsohave the potential to act as a cardiac graft, mending damage to theheart. Recently, cultured Human Skeletal Muscle cells have been used inmicro gravity experiments to study the effects of low gravityenvironments on Human Skeletal Muscle.

Accordingly the ability of specific AARS polypeptides to modulatespecific patterns of genes in HSkMC in a time dependent mannerdemonstrates that these proteins play potentially significant roles inthe processes of myogenesis, as well as diseases and disorders resultingfrom the dysfunction, or deterioration of these processes as well asmuscle cell development or metabolism. Accordingly AARS polypeptideswith the ability to modulate gene transcription in muscle cells havetherapeutic utility in a broad range of diseases including for example,the treatment of metabolic disease, cachexia, various muscle wastingconditions, as well as musculoskeletal diseases.

Methods:

The ability of AARS polypeptides to modulate gene expression is assessedusing a high-throughput microfluidic real-time quantitative PCR(RT-qPCR) approach (Fluidigm Corporation). (See Petriv et al., (2010)PNAS (doi/10.1073/pnas.1009320107) in Human Marrow Stromal Cells (HMSC)and Human Skeletal Muscle Cells (HSkMC). In the experiments reportedhere, Human HSkMC (Cat #150-050 and HMSC (Cat #492-050 were purchasedfrom Cell Applications. HMSC cells are cryopreserved at second passageand can be cultured and propagated to 10 population doublings. Here HMSCin the 6^(th) Passage are used. Human Skeletal Muscle Cells (HSkMC) arecryopreserved at second passage and can be cultured and propagated forat least 15 population doublings. In the experiments reported here HSkMCat passage 6 post harvest from normal human donor are used.

In both cases, cells are plated at 50000 cells/mL in 100 μL, volume ofgrowth media and exposed to AARS polypeptides at a concentration of 250nM, or as otherwise indicated below, for 24 hours and 72 hours. Controlsinclude Differentiation media with a standard cocktail to promote (1)Adipogenesis, (2) Osteogenesis, (3) Chondrogenesis and (4) Skeletalmuscle myotube formation. Additional controls include untreated wellscontaining only growth media. Two wells were run for eachDifferentiation control. Controls: all media was made utilizing DMEM asthe basal media. Standard literature was followed and Differentiationmedia was purchased from Cell Applications. Per the vendor,differentiation media contained the following additives: Skeletal muscledifferentiation cocktail: FBS, insulin, glutamine, FGF, EGF;Adipogenesis cocktail: insulin, dexamethasone and IBMX; Osteogenesiscocktail: FBS, dexamethasone, ascorbate 2 phosphate,beta-glycerophosphate; Chondrogenesis cocktail: insulin,ascorbate-2-phosphate, and TGF-β1.

Standard protocols for using an ABI (Applied Biosystems, Item # AM1728)TAQMAN® Gene Expression Cells-to-CT™ Kit are utilized to lyse cells andharvest genomic material. An ABI Pre-Amp Mix (Applied Biosystems,Item#4391128) is used to initiate pre-amplification. Gene specificprimers are created using a Primer 3 program and purchased from IDTtechnologies. Fluidigm profiling arrays (Item # BMK-M-96.96) were usedfor actual quantitative PCR with standard Fluidigm loading reagents andpipetting devices. Table E1 below lists the genes profiled.

TABLE E1 List of genes assessed in transcriptional profiling CompiledUnique List refseq_nt Full_name_(—) Synonyms ABCA1 NM_005502 ATP-bindingcassette, sub- ABC- family A (ABC1), member 11|ABC1|CERP|FLJ14958|HDLDT1| MGC164864|MGC165011|TGD ACTB NM_001101actin, beta PS1TP5BP1 ACTG1 NM_001614 actin, gamma 1ACT|ACTG|DFNA20|DFNA26 ACVR2B NM_001106 activin A receptor, typeACTRIIB|ActR-IIB|MGC116908 IIB APOA1 NM_000039 apolipoprotein A-IMGC117399 ARNT NM_178427 aryl hydrocarbon receptor HIF- nucleartranslocator 1beta|HIF1B|HIF1BETA|TANGO| bHLHe2 BAD NM_032989BCL2-associated agonist BBC2|BCL2L8 of cell death BCL2 NM_000657 B-cellCLL/lymphoma 2 Bcl-2 BMP2 NM_001200 bone morphogenetic BMP2A protein 2BMP4 NM_130851 bone morphogenetic BMP2B|BMP2B1|MCOPS6|OFC11| protein 4ZYME C3AR1 NM_004054 complement component 3a AZ3B|C3AR|HNFAG09 receptor1 CASP3 NM_032991 caspase 3, apoptosis- CPP32|CPP32B|SCA-1 relatedcysteine peptidase CAV1 NM_001753 caveolin 1, caveolaeBSCL3|CGL3|MSTP085|VIP21 protein, 22 kDa CDH5 NM_001795 cadherin 5, type2 7B4|CD144|FLJ17376 (vascular endothelium) CFLAR NM_003879 CASP8 andFADD-like CASH|CASP8AP1|CLARP|Casper| apoptosis regulatorFLAME|FLAME-1|FLAME1|FLIP| I-FLICE| MRIT|c-FLIP|c-FLIPL|c-FLIPR| c-FLIPSCOMP NM_000095 cartilage oligomeric matrix EDM1|EPD1|MED|MGC131819|protein MGC149768| PSACH|THBS5 CSF1 NM_172212 colony stimulating factor1 MCSF|MGC31930 (macrophage) CTGF NM_001901 connective tissue growthCCN2|HCS24|IGFBP8|MGC102839| factor NOV2 CTNNB1 NM_001904 catenin(cadherin- CTNNB|DKFZp686D02253|FLJ25606| associated protein), beta 1,FLJ37923 88 kDa DAAM1 NM_014992 dishevelled associated FLJ41657|KIAA0666activator of morphogenesis 1 ELN NM_001081755 elastinFLJ38671|FLJ43523|SVAS|WBS|WS ENO1 NM_001428 enolase 1, (alpha)ENO1L1|MPB1|NNE|PPH FABP3 NM_004102 fatty acid binding proteinFABP11|H-FABP|MDGI|O-FABP 3, muscle and heart (mammary-derived growthinhibitor) FAK NM_001199649 focal adhesion kinase fak1 FGF4 NM_002007fibroblast growth factor 4 HBGF-4|HST|HST-1|HSTF1|K-FGF| KFGF FIGFNM_004469 c-fos induced growth VEGF-D|VEGFD factor (vascular endothelialgrowth factor D) FLT1 NM_002019 fms-related tyrosine kinase FLT|VEGFR1 1(vascular endothelial growth factor/vascular permeability factorreceptor) FOXA1 NM_004496 forkhead box A1 HNF3A|MGC33105|TCF3A GAPDHNM_002046 glyceraldehyde-3- G3PD|GAPD|MGC88685 phosphate dehydrogenaseGFAP NM_002055 glial fibrillary acidic FLJ45472 protein SLC2A4 NM_001042solute carrier family 2 GLUT4 (facilitated glucose transporter), member4 HAND1 NM_004821 heart and neural crest Hxt|Thing1|bHLHa27|eHandderivatives expressed 1 HIF1A NM_181054 hypoxia inducible factor 1,HIF-1alpha|HIF1|HIF1- alpha subunit (basic helix-ALPHA|MOP1|PASD8|bHLHe78 loop-helix transcription factor) HK2 NM_000189hexokinase 2 DKFZp686M1669|HKII|HXK2 HMGB1 NM_002128 high-mobility groupbox 1 DKFZp686A04236|HMG1|HMG3| SBP-1 HNF4A NM_178850 hepatocyte nuclearfactor FLJ39654|HNF4|HNF4a7|HNF4a8| 4, alpha HNF4a9|HNF4alpha|MODY|MODY1|NR2A1| NR2A21| TCF|TCF14 HPRT1 NM_000194hypoxanthine HGPRT|HPRT phosphoribosyltransferase 1 HSPB1 NM_001540 heatshock 27 kDa protein 1 CMT2F|DKFZp586P1322|HMN2B| HS.76067|HSP27|HSP28|Hsp25|SRP27 ICAM1 NM_000201 intercellular adhesionBB2|CD54|P3.58 molecule 1 IFNG NM_000619 interferon, gamma IFG|IFI IGF1NM_001111285 insulin-like growth factor 1 IGF-I|IGF1A|IGFI (somatomedinC) IGF2 NM_001127598 insulin-like growth factor 2C11orf43|FLJ22066|FLJ44734|INSIGF| (somatomedin A) pp9974 IGFBP3NM_001013398 insulin-like growth factor BP-53|IBP3 binding protein 3IGFBP5 NM_000599 insulin-like growth factor IBP5 binding protein 5 IKBKBNM_001556 inhibitor of kappa light FLJ33771|FLJ36218|FLJ38368|polypeptide gene enhancer FLJ40509| in B-cells, kinase betaIKK-beta|IKK2|IKKB|MGC131801| NFKBIKB IL10 NM_000572 interleukin 10CSIF|IL-10|IL10A|MGC126450| MGC126451| TGIF IL1B NM_000576 interleukin1, beta IL-1|IL1-BETA|IL1F2 IL3 NM_000588 interleukin 3 (colony-IL-3|MCGF|MGC79398|MGC79399| stimulating factor, MULTI-CSF multiple) IL4NM_172348 interleukin 4 BCGF-1|BCGF1|BSF1|IL- 4|MGC79402 IL5 NM_000879interleukin 5 (colony- EDF|IL-5|TRF stimulating factor, eosinophil) IL6RNM_181359 interleukin 6 receptor CD126|IL-6R-1|IL-6R-alpha|IL6RA|MGC104991 IL8 NM_000584 interleukin 8 CXCL8|GCP-1|GCP1|LECT| LUCT|LYNAP|MDNCF| MONAP|NAF|NAP-1|NAP1 ITGA5 NM_002205 integrin, alpha 5CD49e|FNRA|VLA5A (fibronectin receptor, alpha polypeptide) KDR NM_002253kinase insert domain CD309|FLK1|VEGFR|VEGFR2 receptor (a type IIIreceptor tyrosine kinase) LEP NM_000230 leptin FLJ94114|OB|OBS LPLNM_000237 lipoprotein lipase HDLCQ11|LIPD MAPK11 NM_002751mitogen-activated protein P38B|P38BETA2|PRKM11|SAPK2| kinase 11SAPK2B|p38-2|p38Beta MMP1 NM_002421 matrix metallopeptidase 1 CLG|CLGN(interstitial collagenase) MMP3 NM_002422 matrix metallopeptidase 3CHDS6|MGC126102|MGC126103| (stromelysin 1, MGC126104| progelatinase)MMP-3|SL-1|STMY|STMY1|STR1 MYH1 NM_005963 myosin, heavy chain 1,MGC133384|MYHSA1|MYHa| skeletal muscle, adult MyHC-2X/D| MyHC-2x MYH11NM_022844 myosin, heavy chain 11, AAT4|DKFZp686D10126| smooth muscleDKFZp686D19237| FAA4|FLJ35232|MGC126726|MGC32963| SMHC|SMMHC MYH7NM_000257 myosin, heavy chain 7, CMD1S|CMH1|DKFZp451F047| cardiacmuscle, beta MGC138376| MGC138378|MPD1|MYHCB|SPMD| SPMM MYOD1 NM_002478myogenic differentiation 1 MYF3|MYOD|PUM|bHLHc1 NFATC1 NM_172390 nuclearfactor of activated MGC138448|NF- T-cells, cytoplasmic, ATC|NFAT2|NFATccalcineurin-dependent 1 NFATC2 NM_173091 nuclear factor of activatedNFAT1|NFATP T-cells, cytoplasmic, calcineurin-dependent 2 NFKB1NM_003998 nuclear factor of kappa DKFZp686C01211|EBP-1|KBF1| lightpolypeptide gene MGC54151| enhancer in B-cells 1NF-kappa-B|NF-kappaB|NFKB- p105| NFKB-p50|p105|p50 NOS2 NM_000625 nitricoxide synthase 2, HEP-NOS|INOS|NOS|NOS2A inducible NOTCH1 NM_017617notch 1 TAN1|hN1 NR3C1 NM_001024094 nuclear receptor subfamilyGCCR|GCR|GR|GRL 3, group C, member 1 (glucocorticoid receptor) NRP2NM_201279 neuropilin 2 MGC126574|NP2|NPN2|PRO2714| VEGF165R2 PAX7NM_013945 paired box 7 FLJ37460|HUP1|PAX7B|RMS2 PDGFB NM_033016platelet-derived growth FLJ12858|PDGF2|SIS|SSV|c-sis factor betapolypeptide (simian sarcoma viral (v- sis) oncogene homolog) PDK4NM_002612 pyruvate dehydrogenase FLJ40832 kinase, isozyme 4 PLA2G1BNM_000928 phospholipase A2, group MGC119834|MGC119835|PLA2|PLA2A| IB(pancreas) PPLA2 PLIN1 NM_002666 lipid droplet associated perilipinprotein PPARG NM_138712 peroxisome proliferator-CIMT1|GLM1|NR1C3|PPARG1|PPARG2| activated receptor gamma PPARgamma QARSNM_005051 glutaminyl-tRNA GLNRS|PRO2195 synthetase RHOA NM_001664 rashomolog gene family, ARH12|ARHA|RHO12|RHOH12 member A RUNX1 NM_001754runt-related transcription AML1|AML1-EVI- factor 1 1|AMLCR1|CBFA2|EVI-1|PEBP2aB RXRA NM_002957 retinoid X receptor, alphaFLJ00280|FLJ00318|FLJ16020|FLJ16733| MGC102720|NR2B1 SERPINE1NM_001165413 serpin peptidase inhibitor, PAI|PAI-1|PAI1|PLANH1 clade E(nexin, plasminogen activator inhibitor type 1), member 1 SMAD2NM_005901 SMAD family member 2 JV18|JV18-1|MADH2|MADR2| MGC22139|MGC34440|hMAD-2|hSMAD2 SMAD4 NM_005359 SMAD family member 4DPC4|JIP|MADH4 TERT NM_198255 telomerase reverse EST2|TCS1|TP2|TRT|hEST2transcriptase TGFB1 NM_000660 transforming growthCED|DPD1|LAP|TGFB|TGFbeta factor, beta 1 TGFB3 NM_003239 transforminggrowth ARVD|FLJ16571|TGF-beta3 factor, beta 3 THBS4 NM_003248thrombospondin 4 TSP4 TNF NM_000594 tumor necrosis factorDIF|TNF-alpha|TNFA|TNFSF2 TUBB NM_178014 tubulin, betaM40|MGC117247|MGC16435|OK/SW- cl.56|TUBB1| TUBB5 TUBB1 NM_030773tubulin, beta 1 tubulin isoform beta (1) TUBG1 NM_001070 tubulin, gamma1 GCP-1|TUBG|TUBGCP1 VCAM1 NM_080682 vascular cell adhesionCD106|DKFZp779G2333|INCAM- molecule 1 100|MGC99561 VEGFA NM_003376vascular endothelial MGC70609|MVCD1|VEGF|VPF growth factor A VIMNM_003380 vimentin FLJ36605 WISP1 NM_080838 WNT1 inducible signalingCCN4|WISP1c|WISP1i|WISP1tc pathway protein 1 WNT1 NM_005430wingless-type MMTV INT1 integration site family, member 1

Bioinformatics Analysis:

Data retrieved in .csv format from the Biomark machine by Fluidigm isconverted to a tabular format including sample, mRNA, and replicateinformation along with the raw fluorescence value. PCR reactions thatfailed are marked as missing. Multiple experiments were combined afternormalizing to total expression of mRNA species. All measured mRNAexpression is filtered based on the requirement of detection in at least2 of all of the biological replicates tested. We assessed technical,biological and set deviation mean in entire dataset.

For data analysis Ct values for all genes of interest are firstnormalized to the averaged Ct values for housekeeping genes from thecorresponding sample to obtain ΔCt values (ΔCt=Ct gene−Ct averagehousekeeping genes). Genes from each sample are then normalized to thesame gene in untreated control to obtain ΔΔCt values (ΔΔCt=ΔCt controlsample−ΔCt treated sample).

To obtain fold change values up-regulated genes (i.e. ΔΔCts greater than0) are subject to the following calculation: Fold Change=2̂ΔΔCt. Fordown-regulated genes (i.e. ΔΔCts less than 0): Fold Change=−(2̂|ΔΔCt|).

Cellular Proliferation Assays (Assays A1-A11 in the Data Tables Below)

Background and Therapeutic Relevance:

The ability to modulate the rate of cellular proliferation and apoptosisof different cell types represents a fundamental property of manytherapeutic compounds, and is of direct relevance to the treatment andprevention of a broad range of diseases and disorders.

Accordingly AARS polypeptides with the ability to modulate the rate ofcellular proliferation and or apoptosis have significant therapeuticutility in a broad range of diseases including, as growth factors, anddifferentiation factors for stem cells, and in treatment regimens toenhance or suppress the proliferation of specific cell types of interestin vivo or in vitro, including for example, haemopoietic cells,immunomodulatory cells, cancer, and for the treatment and prevention ofdiseases associated with aging, including for example neurodegeneration,peripheral neuropathy, and loss of muscular and soft tissue tone.

Methods:

Effects of the AARS polypeptides on cellular proliferation is assessedusing one or more of the methods listed below, and as more specificallyelaborated in the methods below.

Hoechst 33432.

Standard cell counts to assess proliferation are performed using Hoechst33432, which is a cell-permeant nuclear counterstain that emits bluefluorescence when bound to dsDNA. It is available as a solution(Invitrogen Cat # H-3570) that is used at a final concentration of 1ug/mL in either media or PBS. Cells are grown in 96 well plates in thepresence of AARS polypeptides for a standard growth time of 48 hours, orlonger depending on cell type and as described in the examples below.

ATP-Lite.

Cellular ATP levels correlate with cellular health and can be readilydetermined using a variety of commercially available kits. ATP-lite(Perkin-Elmer, Cat #6016947 Boston, Mass. 02481) which is a homogenousmixture of lysis solution and ATP-detection reagent. is pre-mixed beforeuse and is used 1:1 v:v ratio with cultured cells. Plates are incubatedfor 5 minutes to promote lysis and plates are measured using aluminescent plate reader. Cells are grown in 96 well plates in thepresence of AARS polypeptides for a standard growth time of 48 hours, orlonger depending on cell type and as described in the examples below.

ALAMARBLUE® (Resazurin) is a cell viability indicator which is based onthe redox state of the cells. Resazurin, the active ingredient, is anontoxic, cell permeable compound that is blue in color and virtuallynonfluorescent when present in its oxidized form. However upon enteringnormal viable cells, resazurin is rapidly reduced to resorufin, whichproduces a red fluorescence signal. Viable cells continuously convertresazurin to resorufin, thereby generating a quantitative measure ofviability—and cytotoxicity. The lack of toxicity allows long-termexposure of cells to resazurin without negative impact; cells grown inthe presence of resazurin were found to produce similar numbers ofviable cells as control cells, as determined by flow cytometricanalysis.

Measurements are made by adding a solution of Resazurin/ALAMARBLUE® tocells, incubating them for 1-4 hours, and reading the fluorescence orabsorbance. The amount of fluorescence or absorbance is proportional tothe number of living cells and corresponds to the cells metabolicactivity. Damaged and nonviable cells have lower innate metabolicactivity and thus generate a proportionally lower signal than healthycells. After incubation with ALAMARBLUE®, samples can readily bemeasured on fluorescence and absorbance instrumentation. Forfluorescence readings: 530 nm excitation and 590 nm emission filtersettings are used.

Cells are grown in 96 well plates in the presence of AARS polypeptidesfor a standard growth time of 48 hours, or longer depending on cell typeand as described in the examples below.

Acetylated LDL Uptake in HepG2C3A Human Hepatocyte Cells. (Assay B1 inthe Data Tables Below)

Background and Therapeutic Relevance:

LDL is the major carrier of cholesterol in the blood, accounting formore than 60% of the cholesterol in plasma. In humans, the hepatic LDLreceptor is responsible for clearing around 70% of plasma LDL fromcirculation. Internalized LDL is degraded to free cholesterol and aminoacids in the lysosome. The liver is the most important organ for LDLcatabolism and LDL receptor activity in humans. LDL that is notinternalized and remains in circulation can be transported byendothelial cells into the vessel wall, resulting in the formation ofatherosclerotic plaques. Circulating LDL can also be taken up bymacrophages and this can also contribute to the formation of plaques.Increasing LDL uptake into hepatic tissue is thought to be beneficial tohuman health and finding safe and efficacious therapeutics that may thepositively regulate this process may provide new therapies forcardiovascular and metabolic diseases. To investigate whether the uniqueproperties of AARS polypeptides can regulate uptake of acetylated LDL, astandard assay for measuring acetylated LDL uptake is employed inHepG2C3a cells.

Accordingly AARS polypeptides with the ability to modulate LDL uptakehave significant therapeutic utility in a broad range of diseasesincluding for example, the treatment of hypercholesteremia,hyperlipidemia, type 1 and 2 diabetes, metabolic syndrome, and vasculardiseases including atherosclerosis

Methods:

HEPG2C3a cells (ATCC# CRL-10741) are maintained in Eagles MinimalEssential (EMEM) medium supplemented with 10% FBS (HyCloneCat#SH30910.03), 50u/mL penicillin/50 μg/mL streptomycin, (Invitrogen)in 15 mL medium in 75 mL flasks. Cells are grown at 37° C., 5% CO₂, in ahumidified environment and utilized in BSL2 certified tissue culturehoods using sterile technique and appropriate personal protectiveequipment including goggles, gloves and lab coats. HEPG2C3a express theLDL-receptor and are competent for acetylated LDL uptake when grown onclear bottom collagen coated plates. A 100 μL volume of cells is platedon collagen coated plates (Invitrogen Cat#A11428) overnight in completemedium (above) at a cell density of 50,000 cells/mL. Cells are washedonce with PBS (Invitrogen Cat#10010) and 80 μL of serum free EMEM isadded to each well. AARS polypeptides at a final concentration of 250 nMper well are added in a consistent volume in sterile PBS to each well. Aunique AARS polypeptide is placed in each well. Cells are serum starvedand exposed to the AARS polypeptides for 16 hours. Following the 16 hourincubation, the, supernatant is collected and soluble ICAM is measuredusing a standard ELISA kit from RND Systems (Cat # DY643), and serumfree media supplemented with 5 μg/mL ac-LDL (Alexa Fluor 488 labeled Cat# L23380, Invitrogen) is added to each well. Following a 2 hourincubation at 37° C. 5% CO₂, cells are washed twice with sterile PBSbefore 100 μL PBS is added to each well for quantification. Plates wereanalyzed for total fluorescent intensity using a bottom read on a VictorX5 fluorescent plate reader (Perkin Elmer) at an excitation wavelengthcentered around 485 nm, and an emission wavelength centered around 535nm. Cells are stained with Hoechst dye and fluorescent intensity 405 nmExcitation/450 nM Emission is read to confirm total cell number isconsistent across the plate.

Regulation of Human Neutrophil Oxidative Burst and Elastase Production(Assays C1-C3 in the Data Tables Below)

Neutrophil Oxidative Burst

Background and Therapeutic Relevance:

Phagocytosis by polymorphonuclear neutrophils and monocytes constitutesan essential arm of host defense against infections by microorganismsincluding bacteria and fungi. The phagocytic process can be separatedinto several major stages: chemotaxis (migration of phagocytes toinflammatory sites), attachment of particles to the cell surface ofphagocytes, ingestion (phagocytosis) and intracellular killing byoxygen-dependent (oxidative burst) and oxygen-independent mechanisms.Reduced or missing burst activity is observed in inborne defects likethe chronic granulomatous disease (CGD). CGD is a heterogeneous group ofinherited disorders that usually manifests itself during the first twoyears of life. The disease is characterized by repeated andlife-threatening infections caused by bacterial and fungal organisms.These infections typically consist of pneumonia, lymphadenitis, orabscesses that involve lymph nodes, lungs, and liver. The NADPH oxidaseis the enzyme system responsible for producing superoxide anion, whichis quickly converted to hydrogen peroxide and hydroxyl radicals.Abnormalities in the constituent peptides of the NADPH oxidase enzymesystem lead to the dysfunctions characteristic of CGD. Neutrophils fromCGD patients fail to produce a significant oxidative burst followingstimulation. Different forms of CGD are described (classical X-linkedCGD and autosomal recessive patterns). The oxidative burst ofgranulocytes is impaired in transplantation, later stages of HIVinfection, and in the elderly, making these populations more susceptibleto secondary infection and exacerbations of inflammatory disease.Various immunomodulators (e.g., cytokines (GM-CSF, G-CSF, TNF) or drugs)also seem to have effects on the oxidative burst. There is the potentialfor proteins with the ability to up-regulate or down-regulate oxidativeburst in a therapeutic fashion to be useful for a variety of differentdisease states.

Methods:

The protein kinase C ligand phorbol 12-myristate 13-acetate (PMA) can beutilized in this assay as an agonist of the oxidative burst process.Heparinized whole blood is mixed with sterile dextran (0.6% finalconcentration) for 1 hour and allowed to separate into layers. The lowerlayer contains neutrophil, monocytes and red blood cells. An ammoniumchloride lysis step is utilized to remove all RBCs and a 97% purepopulation of neutrophils with approximately 3% monocyte contaminationremains following lysis step. Upon stimulation, granulocytes andmonocytes produce reactive oxygen metabolites (superoxide anion,hydrogen peroxide, hypochlorous acid) which destroy bacteria inside thephagosome. Formation of the reactive oxidants during the oxidative burstcan be monitored by the addition and oxidation of Amplex Red. Thepercentage of cells having produced reactive oxygen radicals are thenanalyzed as well as their mean fluorescence intensity using afluorescent plate reader. The typical time course for this reaction is10 minutes, with obvious burst being seen by 2 minutes and a drop off ofsignal being seen by 20 minutes. This assay can be run in agonist modein the absence of PMA or in antagonist mode, with concomitantadministration of AARS polypeptides and PMA at a concentration that isbelow the EC50 for this compound.

Regulation of Human Neutrophil Elastase Production

Background and Therapeutic Relevance:

Neutrophil elastase is a serine protease that has been implicated ashaving a specific role in the development of a wide range of humandiseases, including inflammatory disorders of the lung andcardiovascular system. Although its key physiologic role is in innatehost defense, it can also participate in tissue remodeling and possessessecretagogue actions that are now recognized as important to localinflammatory signals. Neutrophil elastase activity has been implicatedin the development of emphysema for several decades, however onlyrelatively recently has a pathogenetic function been ascribed to thisserine proteinase in situations where excessive extracellular matrixdeposition occurs. The use of genetically manipulated animal models isstarting to uncover the potential ways in which its actions mightinfluence fibrotic lung repair. Emerging evidence suggests that theengagement of cellular pathways with more direct effects on fibrogenicmediator generation and collagen synthesis appears to underpin theactions of neutrophil elastase in promoting lung matrix accumulation.Human neutrophil elastase is also present within atherosclerotic plaqueswhere it contributes to matrix degradation and weakening of the vesselwall associated with the complications of aneurysm formation and plaquerupture. It is joined by other extracellular proteases in these actionsbut the broad range of substrates and potency of this enzyme coupledwith activity associated with neutrophil degranulation single thisdisruptive protease out as therapeutic target in atheroscleroticdisease.

Methods:

This assay uses the ENZCHEK® Elastase Assay Kit (Invitrogen Catalog #E-12056). Neutrophils are prepared from fresh human blood using a 6%dextran solution and red blood cells are lysed before plating cells inRPMI media (media should be un-supplemented with no serum, noantibiotics). A 1.0 mg/mL stock solution of the DQ elastin substrate isprepared by adding 1.0 mL of deionized water (dH2O) directly to one ofthe three vials containing the lyophilized substrate and mixing todissolve. 1× Reaction Buffer is prepared by diluting 6 mL of the 10×Reaction Buffer in 54 mL dH2O. A 100 μg/mL working solution of the DQelastin substrate is prepared by diluting the DQ elastin stock solutiontenfold in 1× Reaction Buffer. Porcine pancreatic elastase stocksolution is prepared by making a 100 U/mL stock solution in dH2O. Toassay for elastase activity, 50 μL of 1× Reaction Buffer is pipette intoeach assay well containing 500,000 neutrophils/mL in a 30 μL volume. 8μL of each AARS polypeptide is added per well, and the sample incubatedfor 20 minutes at 37° C. 50 μL of 100 μg/mL DQ elastin working solutionis added to each well and mixed. Samples are incubated at roomtemperature, protected from light, for 30 minutes. Fluorescenceintensity in a fluorescence microplate reader equipped with standardfluorescein filters (ex 485/Em 535) fluorescence may be measured overmultiple time points.

Binding to Toll-Like Receptors and Activation of NFKB (Assays D1-D4 inthe Data Tables Below)

Background and Therapeutic Relevance:

Macrophages are major players in the innate immune system and express alarge repertoire of different classes of pattern recognition receptors(PRRs), including the family of Toll-like receptors (TLRs) which arepowerful regulators and controllers of the immune response.

Stimulation of TLRs by microbial pathogens and endogenous ligandsinitiates signaling cascades that induce the secretion ofpro-inflammatory cytokines and effector cytokines that direct downstreamadaptive immune responses. Endogenous ligands, as well as microbialcomponents, are recognized by and can activate TLRs, raising thepossibility that these receptors may be critical targets for thedevelopment of new therapies for multiple diseases.

Accordingly AARS polypeptides that modulate TLR receptor activity, havetherapeutic utility in a broad range of diseases and disorders includingfor example, inflammatory diseases and disorders, autoimmune diseases,tissue transplantation/organ rejection, cancer prevention or treatment,the modulation of haematopoiesis and infection.

Measurement of TLR Activation in RAW-BLUE Cells

Mouse macrophages sold under the trademark RAW-BLUE™ cells (Invivogen,Catalog code: raw-sp) express all TLRs except TLR5 and include asecreted embryonic alkaline phosphatase (SEAP) gene which is inducibleby NF-kB and AP-1 transcription factors. Upon TLR stimulation, RAW-BLUE™cells activate NF-kB and/or AP-1 leading to the secretion of SEAP whichis measurable when using SEAP detection medium.

Methods:

RAW-BLUE™ cells are washed twice with PBS, trypsinized and resuspendedin fresh Growth Medium (Growth Medium: DMEM, 4.5 g/l glucose, 10%heat-inactivated fetal bovine serum (30 minutes at 56° C.), 100 mg/mLZEOCIN™ 2 mM L-glutamine). Cells are plated at a concentration of 50,000cells/well in a 96 well plate in a total volume of 100 μL, and AARSpolypeptides, controls, or AARS polypeptides (+LPS) are added to eachwell at the concentrations shown in the experiments outlined below.Cells are incubated at 37° C. in a 5% CO₂ incubator for 18 hours. Onexperimental day 2, SEAP detection medium (QUANTI-BLUE™) (InvivogenCatalog code: rep-qb1) is prepared following the instructions and 120 μLis added per well to a clear flat-bottom 96-well plate, and cellsupernatant is added (20 μL). Samples are incubated at 37° C. for about30 minutes to up to 2 hours. SEAP levels are determined using aspectrophotometer and reading absorbance at 650 nM.

To detect AARS polypeptides that specifically block TLR activation thisassay can be modified to identify potential TLR antagonists. In thiscase AARS polypeptides are added to the cells at a final concentrationof about 250 nM per well, (or as otherwise specified in the Examplesbelow) 1 hour prior to adding 50 ng/mL LPS. Cells are incubated and SEAPdetected as described above. PBS control wells with no LPS or AARSpolypeptide alone added are used to find the basal level of TLRstimulation at the time of the measurement. Control wells are pretreatedwith PBS and known TLR agonists and antagonists. The ratio of thebackground subtracted [PBS plus LPS signal] to [AARS polypeptide plusLPS signal] is used to determine percent antagonism.

Human TLR Screening in Hek293 Cells

Human HEK293 cells are genetically modified and sold under the trademarkHEK-Blue™ TLR cells (Invivogen). The TLR2 and TLR4 versions of this celltype selectively express all TLR2 or TLR4 and include a secretedembryonic alkaline phosphatase (SEAP)reporter gene under the control ofan IFN-beta minimal promoter which is fused to five NF-kB and AP-1transcription factors binding sites. With the use of specific TLR 2 or 4agonists (respectively), HEK-BLUE™ TLR2 and HEK-BLUE™ TLR4 cellsactivate NF-kB and/or AP-1 leading to the secretion of SEAP which ismeasurable when using SEAP detection reagent. The HEK-BLUE™ TLR2 cellsare co-transfected with the LPS co-receptor protein CD14 to enhance TLR2responsiveness and improve signal quality. The parent cell expressesendogenous levels of TLR1, 3, 5, 6 and also NOD1.

Methods:

HEK-BLUE™-TLR2 or HEK-BLUE™-TLR4 cells are washed twice with PBS,trypsinized and resuspended in fresh Growth Medium (Growth Medium: DMEM,4.5 g/L glucose, 10% heat-inactivated fetal bovine serum (30 minutes at56° C.), 100 mg/mL ZEOCIN™, 2 mM L-glutamine). Cells are plated at aconcentration of 50,000 cells/well in a 96 well plate in a total volumeof 100 μL, and AARS polypeptides, controls, or AARS polypeptides (+LPS)are added to each well at the concentrations shown in the experimentsoutlined below. Cells are incubated at 37° C. in a 5% CO₂ incubator for18 hours. On experimental day 2, SEAP detection medium (QUANTI-BLUE™)(Invivogen Catalog code: rep-qb1) is prepared following the instructionsand 120 μL is added per well to a clear flat-bottom 96-well plate, andcell supernatant is added (20 μL). Samples are incubated at 37° C. forabout 30 minutes to up to 2 hours. SEAP levels are determined using aspectrophotometer and reading absorbance at 650 nM. Control wells arepretreated with PBS and known TLR agonists such as UltraPure LPS (TLR-4)or PAM3CSK4 (TLR-2). The ratio of the background subtracted [PBS plusLPS signal] to [AARS polypeptide plus LPS signal] is used to determinepercent agonism.

Cytokine Release (Assays E1-E16 in the Data Tables Below)

Background and Therapeutic Relevance:

Cytokines are a diverse set of small cell signaling protein moleculesthat are used extensively for intercellular communication, and playsignificant roles in normal body homeostasis, including immunomodulationand regulation. Accordingly AARS polypeptides that modulate the release,or biological activities of cytokines, have therapeutic utility in abroad range of diseases and disorders including for example,inflammatory diseases and disorders, autoimmune diseases, tissuetransplantation/organ rejection, cancer prevention or treatment, themodulation of haematopoiesis and infection.

Cytokine Release from Cells in Culture

Methods:

Test cells are seeded into a 24-well plate at density of about 1 millioncells/well in 1 mL of growth media. Cells are treated with either AARSpolypeptide (at the concentrations shown in the examples below) or anequal volume of PBS and incubated overnight at 37° with 5% CO₂.Following cell treatment, samples are centrifuged at 4° C. in a swingingbucket centrifuge at 2,000×g for 5 minutes. Media is carefully removedso as to not disturb the cell pellet and transferred to a new tube.Samples are assayed immediately or snap frozen in liquid nitrogen forsubsequent analysis. Cytokine release (including the cytokines MIF,IL-8, IL-10, Serpin E1, GM-CSF, GRO, IL-1 alpha, IL-1beta, IL-1ra,IL-6., MCP-1, RANTES and TNF-alpha) is determined using commerciallyavailable kits (R&D Systems, Inc, MN, USA) or via a contract researchorganization (MD Biosciences (St. Paul, Minn.).

Cytokine Release from Human Whole Blood

Methods:

Human whole blood is obtained from normal human donors and collectedwith heparin in standard collection tubes. Blood is used on the same dayas it is collected to ensure adequate cell health. Blood is mixed gentlyand plated in an 100 μL volume into 96 well polycarbonate V bottomplates. AARS polypeptides are added and slowly mixed into blood 2× usinga multichannel pipet set on 50 μL. Filter tips are used for allexperimentation and full PPE is worn. All experimentation occurs in adedicated biosafety hood that is suitable for experimentation with humanblood. Blood is incubated overnight at 37° C. with 5% CO₂. Followingcell treatment, samples are centrifuged in a swinging bucket centrifugeat 2,000×g for 5 minutes. Supernatant is collected for cytokine ELISAsELISA are performed as described previously.

Cytokine Release from PBMCs

Methods:

To isolate peripheral blood mononuclear cells freshly isolated humanwhole blood is gently layered over Sigma HISTOPAQUE®-1077 at a ratio of1:1 in 50 mL conical tubes at room temperature. Layered samples arecentrifuged at 400×g in a swinging bucket clinical centrifuge for 30minutes at room temperature with no brake. The white cellular layer atthe interface between the plasma and density gradient is then removed bypipet. These peripheral blood mononuclear cells are washed twice withRPMI-1640 (Invitrogen #22400-105) by dilution and centrifugation for 10minutes at 250×g. The washed PBMC were resuspended in RPMI-1640+10% FBSand plated at 1×10⁶ cells/mL.

Cytokine Release from Human Synoviocytes

Background and Therapeutic Relevance:

A large number of studies have demonstrated that IL-6 and IL-8 areoverproduced in several diseases, and thus may play a fundamental rolein the pathogenesis of inflammatory disease. IL-6 activates endothelialcell production, leading to the release of IL-8 and monocytechemoattractant protein, expression of adhesion molecules, andrecruitment of leukocytes to inflammatory sites. These cytokines areexpressed in cell types associated with inflammatory disease, includingcells involved in the pathogenesis of systemic juvenile arthritis,systemic lupus erythematosus, Crohn's disease, and rheumatoid arthritis.One of the most important systemic actions of cytokine production is theinduction of the acute phase response. Acute phase proteins are producedprimarily by the liver and include proteins that promote the immuneresponse through activation of complement, induction of proinflammatorycytokines, and stimulation of neutrophil chemotaxis. Alternatively, theacute phase response can be helpful, and acute-phase proteins, such asproteinase antagonists, opsonins, and procoagulants, help limit tissuedestruction by resolving inflammation. In particular, IL-6 can stimulatesynoviocyte proliferation and osteoclast activation, leading to synovialpannus formation and repair. IL-6 acts with IL-1 to increase productionof matrix metalloproteinases, which may contribute to joint andcartilage destruction. However, IL-6 may also have protective effects inthe joint, as suggested by the finding that this cytokine induces theexpression of the tissue inhibitor of metalloproteinase and stimulatesproteoglycan synthesis when injected into the joints of mice withantigen-induced arthritis. Human Fibroblast-Like Synoviocytes-RheumatoidArthritis (HFLS-RA) are isolated from synovial tissues obtained frompatients with Rheumatoid Arthritis (RA). They are cryopreserved atsecond passage and can be cultured and propagated at least 5 populationdoublings. HFLS are long known for their role in joint destruction byproducing cytokines and metalloproteinases that contribute to cartilagedegradation.

Accordingly AARS polypeptides with the ability to modulate the growth,differentiation, or cytokine release profile of fibroblast-likesynoviocytes-rheumatoid arthritis (HFLS-RA) have therapeutic utility ina broad range of diseases including for example, the treatment ofinflammatory diseases and disorders including systemic juvenilearthritis, systemic lupus erythematosus, Crohn's disease, and rheumatoidarthritis.

Methods:

HFLS-RA, adult cells (Cell Applications Cat #408RA-05a) are maintainedin Synoviocyte Growth Medium (Cell Applications Cat #415-50) in 15 mLmedium in 125 mL flasks for 1 passage before use. Cells are maintainedat 37° C., 5% CO₂, in a humidified environment and utilized in BSL2certified tissue culture hoods using sterile technique and appropriatepersonal protective equipment including goggles, gloves and lab coats.An 80 μL volume of cells is plated overnight in growth medium at a celldensity of about 50,000 cells/mL. AARS polypeptides at a finalconcentration of 250 nM per well (or as otherwise indicated in theexamples below) are added in sterile PBS to each well followingovernight adherence. Control wells contain untreated cells and areincubated with an equivalent volume of PBS. Cells are exposed toproteins or PBS in basal media (Cell Applications Cat #310-470) for 24hours. Supernatant is removed and IL-8, IL-6 and TNFa ELISA assays arerun according to manufacturer's instructions (RND Systems, Cat # DY206and DY-208, DY-210 Duo-set kits). Proliferation is assessed withResazurin as described previously by adding fresh media containingResazurin to plates following supernatant removal and incubating forthree hours at 37° C. Plates are read on a fluorescent plate reader andviability/proliferation is expressed as a function of resorufinassociated fluorescence of AARS polypeptide treated wells divided byresorufin associated fluorescence of PBS only treated wells.

Human Astrocyte Proliferation and Inflammatory Cytokine Production

Background and Therapeutic Relevance:

Human astrocytes (HA) are derived from human cerebral cortex. They arecryopreserved at second passage and can be cultured and propagated 10population doublings. HA are the most abundant cells in the centralnervous system and they perform many functions such as provision ofmechanical support and nutrients to neurons, and removal of wastes fromneurons. In addition to playing a critical support role for optimalneuronal functioning, they also provide biochemical support ofendothelial cells which form the blood-brain barrier. Recent studieshave shown that astrocytes are capable of regulating neurogenesis byinstructing the stem cells to adopt a neuronal fate and controlling thefunction of single synapses, participate actively in the transfer andstorage of information in the brain. Recognition of the importance ofastrocytes in nervous system functioning is increasing, HA can serve asuseful in vitro model for exploring the diversity of astrocytesfunctions. Astrocytes have been shown to proliferate in response to IL6and TNFalpha. In addition, these cells are capable of making their ownIL6 and TNFalpha. Thus AARS polypeptides which modulate theproliferation and cytokine production in HA have therapeutic utility ina variety of neurological diseases including neuro-inflammation,neurodegeneration, tumorigenesis of the brain, and brain ischemia andrepair.

Methods:

Human Astrocytes (HA) from Cell Applications (Cat #882K-05f) aremaintained in Cell Applications HA Cell Growth Medium (Cat #821-500)according to manufacturer's instructions. Cells are maintained at 37°C., 5% CO₂, in a humidified environment and utilized in BSL2 certifiedtissue culture hoods using sterile technique and appropriate personalprotective equipment including goggles, gloves and lab coats. An 80 μLvolume of cells is plated on collagen coated plates overnight incomplete medium (above) at a cell density of 50,000 cells/mL. Cells arewashed once with PBS and 80 μL of serum free growth media is added toeach well. AARS polypeptides at a final concentration of 250 nM per well(or as otherwise described in the examples below) are added in aconsistent volume in sterile PBS to each well. Cells are exposed to AARSpolypeptides for 48 hours and spent media is removed for cytokineassessment (as described previously). Cells are exposed to proteins orPBS in basal media (Cell Applications Cat #310-470) for 48 hours.Supernatant is removed and IL-8 and IL-6 ELISA assays are run accordingto manufacturer's instructions (RND Systems, Cat # DY206 and DY-208,DY-210 Duo-set kits). Proliferation is assessed with Resazurin asdescribed previously by adding fresh media containing Resazurin toplates following supernatant removal and incubating for three hours at37° C. Plates are read on a fluorescent plate reader andviability/proliferation is expressed as a function of resorufinassociated fluorescence of AARS polypeptide treated wells divided byresorufin associated fluorescence of PBS only treated wells.

Human Lung Microvascular Endothelial Cell (HLMVEC) Proliferation andInflammatory Cytokine Production.

Background and Therapeutic Relevance:

The pulmonary vasculature is of great physiological/pathologicalsignificance. It is now recognized to be a tissue composed ofmetabolically active, functionally responsive cells, that interact withcirculating substrates and formed elements in ways that regulate thecomposition of systemic arterial blood, affect target organ functions,and contribute to thrombosis, hemostasis and immune reactions, as wellas tumor metastasis. Human lung microvascular endothelial cells (HLMVEC)exhibit elevated expression of chemoattractant cytokines and celladhesion molecules that provide critical cues for directed migration ofleucocytes into the lung during acute lung injury. This primary celltype can be useful tool for studying various aspects of pathology andbiology of the pulmonary microvasculature in vitro. Alteration in thestructure and function of the microvasculature in response toinflammatory stimuli is believed to be a key factor in organ damage andunder appropriate conditions, may provide a stimulus for repair. Asignificant cause of these vascular alterations is the induction of aninflammatory reaction involving leukocyte infiltration. A variety ofstudies focused on granulocyte adhesion to the endothelium have revealedthat leukocyte recruitment and emigration involves a well-orchestratedadhesion cascade. The adhesion cascade begins when the granulocyteattaches to the endothelium and begins to roll in the direction of fluidflow at a low velocity. As the granulocyte rolls, it becomes activated,subsequently firmly adheres to the endothelium, and migrates across theendothelium into the extravascular space. These adhesion events aremediated, in part, by molecular interactions that occur between CAMs onthe surface of the granulocytes and cognate glycoproteins present on theendothelium. A variety of studies have revealed that the endothelialcell adhesion molecule E-selectin can interact with SLex-type glycanpresenting granulocyte ligands to mediate the attachment and rollingsteps of the adhesion cascade. The downstream steps of the cascadeinvolve the interaction of endothelial-expressed intercellular adhesionmolecule with granulocyte-expressed CD18 integrins.

Thus AARS polypeptides which modulate proliferation and/or cytokineproduction of human lung microvascular endothelial cells havetherapeutic utility in a variety of vascular and pulmonary diseasesincluding inflammatory and obstructive lung diseases including forexample, pulmonary hypertension, chronic obstructive pulmonary disease,idiopathic pulmonary fibrosis, and asthma.

Methods:

HLMVEC (Cell Applications, Catalog #540-05) are maintained in CellApplications Microvascular Endothelial Cell Growth Medium (Cat#111-500), For appropriate growth, an Attachment Factor Solutioncontaining collagen (Cell Applications, Catalog #123-100), is used tocoat plates and flasks before plating cells. Cells are maintained at 37°C., 5% CO₂, in a humidified environment and utilized in BSL2 certifiedtissue culture hoods using sterile technique and appropriate personalprotective equipment including goggles, gloves and lab coats. A 80 μLvolume of cells is plated on collagen coated plates overnight incomplete medium (above) at a cell density of 50,000 cells/mL. Cells arewashed once with PBS and 80 μL, of serum free growth media is added toeach well. AARS polypeptides at a final concentration of 250 nM per well(or as otherwise described in the examples below) are added in aconsistent volume in sterile PBS to each well. Cells are exposed to AARSpolypeptides for 48 hours and spent media is removed for ELISA for celladhesion molecules and cytokine assessment (as described previously).Cell adhesion molecules including soluble VCAM and/or ICAM are measuredusing a standard ELISA kit from RND Systems (Cat # DY643 and DY720respectively). Proliferation is assessed with Resazurin as describedpreviously by adding fresh media containing Resazurin to platesfollowing supernatant removal and incubating for three hours at 37° C.Plates are read on a fluorescent plate reader andviability/proliferation is expressed as a function of resorufinassociated fluorescence of AARS polypeptide treated wells divided byresorufin associated fluorescence of PBS only treated wells.

Cell Adhesion ((Assays F1-F7 in the Data Tables Below)

Background and Therapeutic Relevance:

Cell Adhesion Molecules (CAMs) are proteins located on the cell surfacewhich are involved with the binding with other cells or with theextracellular matrix (ECM) in the process called cell adhesion. Theseproteins are typically transmembrane receptors and are composed of threedomains: an intracellular domain that interacts with the cytoskeleton, atransmembrane domain, and an extracellular domain that interacts eitherwith other CAMs of the same kind (homophilic binding) or with other CAMsor the extracellular matrix (heterophilic binding). Most of the CAMsbelong to four protein families: Ig (immunoglobulin) superfamily (IgSFCAMs), the integrins, the cadherins, and the selectins. Theimmunoglobulin superfamily (IgSF) cell adhesion molecules arecalcium-independent transmembrane glycoproteins, including: neural celladhesion molecules (NCAMs), intercellular cell adhesion molecules(ICAMs), vascular cell adhesion molecule (VCAM), platelet-endothelialcell adhesion molecule (PECAM-1), endothelial cell-selective adhesionmolecule (ESAM), junctional adhesion molecule (JAMs), nectins, and othercell adhesion molecules.

Cell adhesion molecules are cell surface glycoproteins that are criticalfor leukocyte adhesion to the sinusoidal endothelium and transmigrationand cytotoxicity in a variety of inflammatory liver diseases. ICAM-1plays an important role in inflammation, and the increased expression ofICAM-1 on endothelial cells is reflected in the activation ofendothelial cells. ICAM-1 is of particular importance since it mediatesfirm endothelial adhesion and facilitates leukocyte transmigration.Studies have shown that there is an upregulation of ICAM-1 on bothsinusoidal cells and hepatocytes in inflammatory liver conditions suchas hepatitis B viral infection, autoimmune liver disorders, alcoholichepatitis, and liver allograft rejection.

Thus AARS polypeptides which modulate cell adhesion molecule productionand cell adhesion to endothelial cells have therapeutic utility in avariety of inflammatory diseases including for example, cardiovasculardiseases, atherosclerosis, autoimmunity and pulmonary hypertension.

Methods:

Human umbilical vein cells (ATCC, Cat # CRL-2873) (HUVEC) are seeded ata concentration of about 1.2×10⁵ cells/well in 12 well plates coatedwith human fibronectin attachment solution in the suggested ATCC mediaand supplements and grown according to manufacturer's instructions.Cells are stimulated with AARS polypeptides at the indicatedconcentrations, or PBS alone, and incubated overnight in growth media.Human acute monocytic leukemia (THP-1 (TIB-202)), cells are resuspendedinto 0.1% BSA/RPMI serum free medium with calcein AM (6 μL/mL;Invitrogen Cat # C1430) and incubated for 30 minutes. Labeled cells arecollected and resuspended in RPMI medium containing 10% FBS, and thedensity adjusted to 2×10⁶ cells/mL.

100 μL (2×10⁵) labeled THP-1 cells are placed into each well of theHUVEC monolayer in 1 mL of growth media and incubated for 15 minutes.The wells are washed twice with PBS to remove unbound cells, and thenthe cells are read by fluorescent plate reader with an Excitationwavelength of 488 nm and an Emission wavelength of 530 nm.

Cellular Differentiation (Assays G1-G4 in the Data Tables Below)

Adipocyte Differentiation and Proliferation in Primary HumanPre-Adipocyte Cells.

Background and Therapeutic Relevance:

Both obesity and lipodystrophy are commonly associated with pathologiesincluding diabetes and cardiovascular diseases. It is now recognizedthat adipose tissue is an endocrine organ that secretes a wide varietyof factors, and dysregulated secretion affects adipogenesis as well aswhole-body glucose/insulin homeostasis. Excess adipose tissue leading toobesity has become a severe public health threat. Adipose tissuedevelopment can be affected by genetic background, hormonal balance,diet, and physical activity. Adipose tissue mass can increase when fatcells are increased in size due to higher triacylglycerol accumulation.In addition, an increase in fat cell number, arising fromdifferentiation of precursor cells into adipocytes, can also occur evenin adults as observed in severe human obesity and in rodents fed ahigh-carbohydrate or high-fat diet. Adipocytes specifically are thoughtto arise from mesenchymal cells that undergo the commitment anddifferentiation process, adipogenesis. Pre-adipocyte cell lines canundergo adipocyte differentiation upon treatment with adipogenic agentscomprised of synthetic glucocorticoid, dexamethasone (DEX),isobutylmethylxanthine (IBMX), and insulin, have been valuable in thesestudies. Peroxisome proliferator-activated receptor γ (PPARγ) and CCAATenhancer-binding protein (C/EBP) family of transcription factors havebeen firmly established to play critical roles in adipocytedifferentiation. Early during adipocyte differentiation, C/EBPβ andC/EBPδ are induced by DEX and IBMX, respectively, which together theninduce PPARγ and C/EBPα to activate various adipocyte markers that arerequired for adipocyte function. Other transcription factors have alsobeen reported to either positive or negatively regulate adipogenesis andvarious growth factors and hormones can affect adipocyte differentiationby regulating expression of adipogenic transcription factors. In fact,in addition to being the main site for energy storage in mammals bystoring triacyglycerol and releasing fatty acids in times of need,adipose tissue secretes a wide array of molecules that are involved indiverse physiological processes including immune response, vascularfunction, and energy homeostasis. Cytokines such as TNF-α and IL-6 aresecreted from adipocytes. Some of these factors may also affect growthand development of adipose tissue by autocrine/paracrine action.

Thus AARS polypeptides which have the ability to modulate thedifferentiation and/or proliferation of normal human pre-adipocytes havetherapeutic utility in a broad range of diseases including for example,the treatment and prevention of metabolic disease, cardiovasculardiseases, obesity and lipodystrophies, as well as the long termcomplications of diabetes.

Methods:

HPAd (human pre-adipocytes) (Cell Application Cat #803sD) are maintainedaccording to vendor instructions. For culturing, cells are thawedquickly, and transferred immediately into 15 mL of Adipocyte GrowthMedium (Cell Application Cat #811M-250) and plated into a standardsterile tissue culture treated flask. Media is replaced with freshAdipocyte Growth Medium every other day until cell is >60% confluent.Cells are grown at 37° C., 5% CO₂, in a humidified environment andutilized in BSL2 certified tissue culture hoods using sterile techniqueand appropriate personal protective equipment including goggles, glovesand lab coats. Cells are plated in clear bottom black walled 96 welltissue culture treated assay plates for differentiation at aconcentration of about 50,000 cells/mL. AARS polypeptides at a finalconcentration of 250 nM per well (or as otherwise indicated in theExamples below) are added to each assay well. All cells are maintainedin growth media for 2 days with the exception of the positive controlswhich are stimulated with adipogenic differentiation media (CellApplications Cat #811D-250). Cells are exposed to AARS polypeptides for48 hours. Cell adhesion molecules including soluble VCAM and/or ICAM aremeasured using a standard ELISA kit from RND Systems (Cat # DY643 andDY720 respectively). Proliferation is assessed with Resazurin asdescribed previously by adding fresh media containing Resazurin toplates following supernatant removal and incubating for three hours at37° C. Plates are read on a fluorescent plate reader andviability/proliferation is expressed as a function of resorufinassociated fluorescence of AARS polypeptide treated wells divided byresorufin associated fluorescence of PBS only treated wells. Fresh mediais added and differentiation is maintained for 16 days post initialmedia exchange, with fresh media exchanged every other day to maintaincell health. On day 15, cells are placed in serum free media. On day 16,differentiation to mature adipocytes is assessed with Nile Red(Invitrogen, concentration of 3 μM final) staining and quantified with afluorescent plate reader with the appropriate wavelengths. To performthis assay cells are fixed with 10% paraformaldehyde, washed in PBS andpermeabilized in PBS containing 0.5% BSA and 0.1% Triton X-100. Cellproliferation is assessed with an intensity measurement on a fluorescentreader with Hoechst dye 33432 at a concentration of 1 ug/mL final, asdescribed previously. Adipogenesis is expressed as intensity of Nile Redsignal. Hoechst dye signal is used to assess cellular number.

Human Skeletal Muscle Cell Differentiation and Proliferation.

Background and Therapeutic Relevance:

The development of skeletal muscle is a multistep process that involvesthe determination of pluripotential mesodermal cells to give rise tomyoblasts, withdrawal of the myoblasts from the cell cycle anddifferentiation into muscle cells, and finally growth and maturation ofskeletal muscle fibers. Skeletal muscle differentiation involvesmyoblast alignment, elongation, and fusion into multinucleate myotubes,together with the induction of regulatory and structural muscle-specificgenes. At the molecular level, myogenic commitment and muscle-specificgene expression involve the skeletal muscle-specific helix-loop-helix(bHLH) MyoD family of proteins, which includes MyoD, myogenin, myf-5,and MRF4, and the myocyte enhancer-binding factor 2 (MEF2). The DNAbinding activity of MyoD family proteins is attenuated by Id, whichforms complexes with E2a gene products in proliferating cells and isdown-regulated when they are induced to differentiate. The decision todifferentiate into myotubes is influenced negatively by several factors.Treatment of myoblasts with fetal bovine serum, basic fibroblast growthfactor 2, or transforming growth factor (31 is known to inhibitdifferentiation of myoblasts. Myogenesis is also regulated negatively byoncogenes such as c-myc, c-jun, c-fos, H-ras, and E1a. There is verylittle information regarding the signaling that is triggered in themyoblast upon serum withdrawal which leads to the induction of the MyoDfamily gene expression and to muscle differentiation. Myogenicdifferentiation appears to depend on the activation of integrins presenton the plasma membrane of myoblasts suggesting the operation of an“outside-in” biochemical pathway in which integrin is the upstreammolecular species. Interactions of insulin-like growth factor (IGF)-Iand -II with their receptors are also positive regulators of skeletalmuscle differentiation.

Accordingly AARS polypeptides with the ability to modulate muscledevelopment have therapeutic utility in a broad range of diseasesincluding for example, the treatment of metabolic disease, cachexia,various muscle wasting conditions, as well as musculoskeletal diseasewhere muscle atrophy plays a key role in the pathogenesis andsymptomology. Human Skeletal Muscle Cells (HSkMC) can undergodifferentiation to exhibit actin and myosin myofilaments. HSkMC havebeen used in the study of genetic muscular diseases such as MalignantHyperthermia. HSkMC also have the potential to act as a cardiac graft,mending damage to the heart, and thus AARS polypeptides with the abilityto modulate muscle development also have utility as in vitro and in vivoregulators of myogenesis.

Methods:

To assess the potential role of AARS polypeptides in this process, astandard assay of skeletal muscle cell differentiation was employed. Forthis assay, Human Adult Skeletal Muscle Cells (HSkMC, Cell ApplicationCat #150-050 are isolated from healthy human donors from limbal skeletalmuscle. Cells are maintained in HSkMC Growth Medium (Cell Applications,Cat #151-500). These cells can be cultured and propagated for at least15 population doublings. For differentiation, cells are maintained ingrowth media for one passage and then plated at 50,000 cells per mLmedia in to 96 well clear bottom black walled TC treated plates treatedwith collagen at 100 μL per well. Cells are allowed to adhere overnight.AARS polypeptides in PBS, or PBS alone, is added to each well at a finalconcentration of 250 nM protein (or as otherwise indicated in theexamples below). Control wells received the same volume ofDifferentiation Media (Cell Applications Cat #151D-250) at this time.Cells are incubated with protein or differentiation media for 48 hours.At 48 hours, cell culture supernatant is collected from all wells anddifferentiation media is added at a volume of 150 μL to the entire platewith the exception of control wells which are maintained in growth mediaonly. Supernatant is utilized to assess cytokine production includingIL6 and IL8 as described previously. Proliferation is assessed withResazurin as described previously by adding fresh media containingResazurin to plates following supernatant removal and incubating forthree hours at 37° C. Cells are monitored under the microscope and mediais exchanged for fresh Differentiation media every 2 days. On Day 10,media is removed and cells are fixed with 10% paraformaldehyde for 30minutes. Cells are permeabilized with 0.1% Triton X-100 in PBS for 15minutes and cells are stained with TR-Labeled phalloidin and Hoechst33432 (as described previously) to define actin and nuclei respectively.Nuclear intensity is used to determine cell proliferation in each welland phalloidin intensity is used to determine total actin content. Cellsare also stained with alpha actin skeletal muscle antibody (GenTex Cat #GTX101362). Digital photos using a fluorescent microscope as well asvisual inspections and scoring are made of all wells.

Human Bone Marrow Mesenchymal Stem Differentiation and Proliferation.

Background and Therapeutic Relevance:

Mesenchymal stem cells (MSCs) are multipotent stem cells that candifferentiate into a variety of cell types, including osteoblasts,chondrocytes, myocytes, adipocytes, beta-pancreatic islets cells, andpotentially, neuronal cells. Many different events contribute to thecommitment of the MSC to other lineages including the coordination of acomplex network of transcription factors, cofactors and signalingintermediates from numerous pathways. MSCs are of intense therapeuticinterest because they represent a population of cells with the potentialtreat a wide range of acute and degenerative diseases.

Moreover AARS polypeptides with the ability to modulate thedifferentiation of MSCs into different developmental pathways havesignificant therapeutic utility to enable the in vitro or in vivomodulation of hematopoiesis, neurogenesis, myogenesis, osteogenesis, andadipogenesis, as well as in a broad range of disorders and diseases,including for example inflammatory responses, autoimmunity, cancer,neuronal degeneration, muscular dystrophy, osteoporosis, andlipodystrophy. Human MSCs are immuno-privileged, and represent anadvantageous cell type for allogenic transplantation, reducing the risksof rejection and complications of transplantation. Recently, there havealso been significant advances in the use of autologous mesenchymal stemcells to regenerate human tissues, including cartilage and meniscus,tendons, and bone fractures. Many studies have also investigated the useof MSCs for gene therapy, including transplantation of MSCs transfectedwith vascular endothelial growth factor for the improvement of heartfunction after MI in rats, MSCs as vehicles for interferon-β deliveryinto tumors in mice and gene therapy with MSCs expressing BMPs topromote bone formation. Accordingly due to the intense interest as MSCsas direct and modified therapeutics, as well as the potential of AARSpolypeptides to act as therapeutic agents to regulate thedifferentiation of MSCs in vivo, AARS polypeptides were tested aspotential inducers of MSC proliferation and differentiation.

Methods:

hMSC (human marrow stromal cells) (Cell Application Cat #492-050 aremaintained according to vendor instructions. For culturing, cells arethawed quickly, and transferred immediately into 15 mL of Marrow Stromalcell Growth Medium (Cell Application Cat #419-500) and plated into astandard sterile tissue culture treated flask. Media is replaced withfresh Marrow Stromal cell Growth Medium every other day until cellsare >60% confluent. Cells are grown at 37° C., 5% CO₂, in a humidifiedenvironment and utilized in BSL2 certified tissue culture hoods usingsterile technique and appropriate personal protective equipmentincluding goggles, gloves and lab coats. Cells are plated in clearbottom black walled 96 well tissue culture treated assay plates fordifferentiation at a concentration of 50,000 cells/mL. tRNA synthetasederived proteins at a final concentration of 250 nM per well (or asotherwise specified in the Examples below) are added to each assay well.All cells are maintained in growth media for 2 days with the exceptionof the positive controls, which was stimulated with osteogenic orchonodrogenic differentiation media (StemPro, Invitrogen, Cat #A10072-01 and A10071-01 respectively). Cells are exposed to AARSpolypeptides for 48 hours. Soluble VCAM is measured using a standardELISA kit from RND Systems (Cat # DY643). Proliferation is assessed withResazurin as described previously by adding fresh media containingResazurin to plates following supernatant removal and incubating forthree hours at 37° C. Plates are read on a fluorescent plate reader andviability/proliferation is expressed as a function of resorufinassociated fluorescence of AARS polypeptide treated wells divided byresorufin associated fluorescence of PBS only treated wells. Followingan assessment of cell viability, resazurin is removed with two mediaexchanges and 0.5× differentiation media is added to all wells.Differentiation is monitored by visual inspections of all wells for 10days post media exchange, with fresh media exchanged every other day tomaintain cell health. Differentiation was assessed with alkalinephosphatase staining using ELF-97 stain (Invitrogen Cat# E6601) at day10 post first differentiation exchange. (Yang et al, Nature Protocols(6) 187-213 (2011) doi:10.1038/nprot.2010.189).

Human Pulmonary Artery Smooth Muscle Cell (hPASMC) Proliferation andDifferentiation.

Background and Therapeutic Relevance:

Pulmonary artery smooth muscle cells (PASMCs) in normal human adult lungblood vessels are mostly quiescent, non-migratory and are largelycommitted to executing their contractile function in the lung. However,PASMCs are not terminally differentiated and possess the ability tomodulate their phenotype and exit their quiescent state in response tochanging local environmental cues. This differentiation state may occurin development, tissue injury, and vessel remodeling in response tochanges in tissue demand. Pulmonary hypertension (PH) is associated witha variety of underlying conditions including an increase in peripheralpulmonary vascular resistance as a result of increased vascular tone andPASMC contractility and vascular remodeling. Vascular remodelinginvolves PASMC growth, synthesis of matrix material, and alterations incell-cell and cell-matrix interactions in the walls of small pulmonaryarteries (PAs), which lead to increased thickness of the smooth musclecomponent of the vessel wall and abnormal muscularization of thenormally nonmuscularized, distal PAs. This process contributes toreduced lumen diameter and increased peripheral resistance. Although theprecise role of the PASMCs in the initial cause of the disease iscontroversial, the changes that occur play a key role in the clinicalconsequences of the disease. A crucial step in studying cellulardifferentiation is identifying a set of cell-specific or cell-selectivegenes that contribute to the differentiated function(s) of the cell. Avariety of smooth muscle cell (SMC) genes have been identified thatserve as useful markers of the relative state of differentiation ormaturation of the vascular SMCs, such as SM alpha-actin, SM MHC,hl-calponin, SM22-alpha, desmin, metavinculin, smoothelin and others.The most widely used marker is SM alpha-actin, partially because of thecommercial availability of a number of very high-affinity and highlyselective antibodies for this protein. Whether changes in PASMCs resultfrom their inherent characteristics or from dysregulation of molecularevents that govern PASMC growth remains an open question. Howeverdetermining the regulatory cues and managing potential dis-regulationprovides significant therapeutic insight to managing a variety ofvascular and pulmonary diseases including pulmonary hypertension,vascular diseases.

Thus AARS polypeptides which have the ability to modulate thedifferentiation and/or proliferation of normal human PASMCs derived fromadult humans have therapeutic utility in a variety of vascular andpulmonary diseases including inflammatory and obstructive lung diseasesincluding for example, pulmonary hypertension, chronic obstructivepulmonary disease, idiopathic pulmonary fibrosis, and asthma.

Methods:

HPASMC (Cell Applications Cat #352-05a) are maintained in HPASMC growthmedia (Cell Applications Cat #352-05a) in 15 mL medium in 125 mL flasksfor 1 passage before use. Cells are maintained at 37° C., 5% CO₂, in ahumidified environment and utilized in BSL2 certified tissue culturehoods using sterile technique and appropriate personal protectiveequipment including goggles, gloves and lab coats. An 80 μL, volume ofcells is plated on collagen coated overnight in growth medium at a celldensity of 50,000 cells/mL. AARS polypeptides were added in sterile PBSto each well at a final concentration of 250 nM (or as otherwisespecified in the Examples below). Control wells held only an equivalentvolume of PBS. Positive control samples were incubated with vendorsupplied HPASMC differentiation media (Cell Applications Cat #311D-250).Cells are exposed to AARS polypeptides or PBS in basal media (CellApplications Cat #310-470) for 48 hours followed by a media exchange todifferentiation media for the entire plate. Supernatant is collected andutilized to assess cytokine production including IL6 and IL8 asdescribed previously. Proliferation is assessed with Resazurin asdescribed previously by adding fresh media containing Resazurin toplates following supernatant removal and incubating for three hours at37° C. Cells are monitored for 10 days with a media exchange every otherday. Differentiation is assessed after fixation as described above, andpermeabilzation with 0.1% Triton X-100, by quantifying staining tosmooth muscle actin-alpha staining using an anti-SMA-alpha antibody(GeneTex Cat #GTX101362) and an Alexa 405 conjugated secondary antibody.Proliferation is assessed with Hoechst staining after cell fixation in10% formaldehyde for 30 minutes. Hoechst dye is read using a bottomreading fluorescent plate reader with an excitation wavelength (Ex) of405 nm, and an emission wavelength (Em) of 450 nm. Total actin stainingis assessed via the use of an Alexa-488 labeled phalloidin stain(Invitrogen Cat# A12379).

Analysis of the Binding of AARS Polypeptides to Cells (Assays H1-H10 inthe Data Tables Below)

Background and Therapeutic Relevance:

The binding of AARS polypeptides to specific cell types demonstratesthat the cell type in question expresses specific receptors for the AARSpolypeptide in question. Depending upon the cell type in question, cellbinding implies a potential role for the AARS polypeptide in regulatingthe activity or behavior of the cell, or similar types of cell, in vivo.Specific examples of such regulatory roles include for example, thebinding and modulation of B-cells and T-cells(immunomodulation/chemotaxis/autoimmunity/inflammation); HepG2 cells(control of metabolism, cholesterol uptake or metabolism); THP-1,jurkat, Raji cells(immunomodulation/chemotaxis/autoimmunity/inflammation), platelets(thrombopoiesis), 3T3L1 adipocytes (lipogenesis/metabolism), and C2C12mouse myoblasts (myogenesis, osteogenesis).

Binding to Blood Cells

Methods:

Blood is collected in EDTA tubes from healthy donors. 2 mL whole bloodis placed into 5 mL Falcon FACS tube. 2 mL of staining buffer (PBS+2%FBS) is added, vortexed 3-5 seconds, centrifuged for 5 minutes at 300×g.The supernatant aspirated, the wash repeated, and the pellet resuspendedin 2 mL of staining buffer.

100 μl of washed blood is transferred to clean 5 mL FACS sample tubes.His6- or V5-His6-tagged AARS polypeptides are added to tubes at theconcentrations indicated in the specific experiments outlined below andincubated on ice for 45 minutes. After incubation, antibodies for thedifferent cell type surface markers (BD Pharmigen Cat Nos. 560910,555398, 555415, 340953, 560361), and FITC labeled anti-V5 tag antibody(V5-FITC, Invitrogen Cat # R96325) or FITC labeled anti-His6 antibody(AbCam Cat #ab1206) are added to tubes, incubated in the dark on ice 30minutes. After incubation 2 mL of BD FACS Lysing Solution (cat #349202)was added to tubes. Samples are vortexed, and placed on ice for 15minutes. Samples are washed with 1×2 mL PBS and resuspended in 2 mL of2% formaldehyde in PBS prior to FACS analysis. AARS polypeptides thatbind greater than 25% of a cellular population, where antibody alone hasno significant signal, is deemed a hit.

Platelet Binding Assays:

50 μL of washed blood is transferred to clean 5 mL FACS sample tubes,His6- or V5-His6-tagged AARS polypeptides are added to tubes at theconcentrations indicated in the specific experiments outlined below andtubes are placed on ice for 45 minutes. 20 μL CD61 pan platelet antibody(BD Pharmigen, Cat #555754) and 0.5 μL anti-V5-FITC labeled antibody(Invitrogen, R96325) or FITC labeled anti-His6 antibody (AbCam Cat#ab1206) are added to each tube. Tubes are placed on ice and protectedfrom light for 30 minutes. Samples are brought up to a total volume in 2mL of 1% formaldehyde in PBS and analyzed by flow cytometry within 24hours. AARS polypeptides that bind greater than 25% of a cellularpopulation, where antibody alone has no significant signal, is deemed ahit.

Binding to Cells in Culture:

Approximately 1×10⁶ cells in 100 μL complete RPMI medium are placed into5 mL FACS tubes. His6- or V5-His6-tagged AARS polypeptides are added totubes at the concentrations indicated in the specific experimentsoutlined below and tubes are placed on ice for 45 minutes. Cell samplesare washed twice with 1 mL staining buffer (PBS+2% FBS), and then 0.5 μLof anti-V5-FITC antibody (Invitrogen R96325) or FITC labeled anti-His6antibody (AbCam Cat #ab1206) in staining buffer with 200 μg/mL humanIgG, is added and the samples incubated on ice, protected from light,for 30 minutes. Samples are washed twice with 1 mL staining buffer, andthen brought up to a total volume in 2 mL of 1% formaldehyde in PBS andanalyzed by flow cytometry within 24 hours. AARS polypeptides that bindgreater than 25% of a cellular population, where antibody alone has nosignificant signal, is deemed a hit.

Animal Studies: Modulation of Haematopoiesis and Circulating Cytokines

Background and Therapeutic Relevance:

Hematopoiesis (alternatively haemopoiesis or hemopoiesis) is theformation of blood cellular components. All cellular blood componentsare derived from haematopoietic stem cells (HSCs) which reside in themedulla of the bone (bone marrow) and have the unique ability to giverise to all of the different mature blood cell types. HSCs are selfrenewing: when they proliferate, at least some of their daughter cellsremain as HSCs, so the pool of stem cells does not become depleted. Theother daughters of HSCs (myeloid and lymphoid progenitor cells), howevercan each commit to any of the alternative differentiation pathways thatlead to the production of one or more specific types of blood cells, butcannot themselves self-renew. A change in the blood components inresponse to exposure to an AARS polypeptide therefore suggests that theAARS polypeptide is capable of modulating hematopoiesis, and regulatingthe development of haematopoietic stem cells.

All blood cells can be divided into three lineages; Erythroid cells,lymphocytes and myelocytes.

Erythroid cells are the oxygen carrying red blood cells. Bothreticulocytes and erythrocytes are functional and are released into theblood. Accordingly a reticulocyte count estimates the rate oferythropoiesis, and a change in red blood cell count suggests that anAARS polypeptide modulates erythropoiesis.

Lymphocytes are the cornerstone of the adaptive immune system. They arederived from common lymphoid progenitors. The lymphoid lineage isprimarily composed of T-cells and B-cells (types of white blood cells).Accordingly a change in white blood cell count or composition inresponse to exposure to an AARS polypeptide suggests that that the AARSpolypeptide modulates lymphopoiesis.

Myelocytes, which include granulocytes, megakaryocytes and macrophages,and are derived from common myeloid progenitors, are involved in avariety of roles, including innate immunity, adaptive immunity, andblood clotting. Accordingly a change in myeloid cell count orcomposition in response to exposure to an AARS polypeptide suggests thatthat the AARS polypeptide modulates myelopoiesis. The same rationale canbe used to establish whether the AARS polypeptides modulategranulopoiesis, by measuring changes in granulocyte number in responseto exposure to the AARS polypeptides. A role for the AARS polypeptide inmodulating megakaryocytopoiesis may be inferred by a change inmegakaryocyte or platelet composition or number in the blood.

Cytokine release in either wild type mice, or in various animal modelsystems of inflammation, provides an initial assessment of the potentialability of the AARS polypeptides to modulate inflammatory responses. Therole of AARS polypeptides in modulating acute chronic inflammatoryprocesses for example, can be readily assessed using a mouse model ofdiet induced obesity (DIO). The DIO model centers upon placing rodentson a high fat diet for several months leading to increased obesity,insulin resistance and immune system dysfunction. A particularconsequence of this immune system dysregulation results in increasedproduction of proinflammatory cytokines in DIO animals leading to acondition of chronic systemic inflammation. There is a growing body ofevidence suggesting that low grade inflammation contributes to thedevelopment and maintenance of obesity and a diabetic phenotype that issimilarly observed in the human condition termed metabolic syndrome. Assuch, the ability of AARS polypeptides to modulate the immune system andrestore homeostatic balance towards a resolution of this chronicinflammatory state would be particularly beneficial in numerous diseasesand disorders including but not limited to the treatment and preventionof the symptoms and side effects of metabolic disease, diabetes,cardiovascular diseases, atherosclerosis, obesity, as well as variousautoimmune diseases and disorders, including for example, multiplesclerosis, vascular and allergic disorders.

Methods:

Male wild type control (C57BL/6) or diet induced obesity mice(C57BL/6NHsd) are purchased from Harlan (Indianapolis, Ind.) and housedindividually. DIO mice are fed a high fat diet (Cat. #TD.06414-60% kcalfrom fat) and control mice are fed a normal diet (Cat. #2018S-18% kcalfrom fat). DIO mice are placed on the high fat diet starting at 6 weeksof age for a total of 10 weeks. Both DIO and control mice are allowed tofeed and drink ad libitum. At 16 weeks of age, mice are sorted andrandomized into groups of 5 animals based on weight. On day 2, mice areweighed and tail vein bled (1004) for pre-treatment complete blood count(CBC) analysis. On day 1, mice are weighed and intravenously injectedvia the tail vein with vehicle (PBS) or individual AARS polypeptides at10 mg/kg. Four hours post-injection, mice are facial vein bled(150-2004) for subsequent cytokine analysis. On days 2, 3, & 4, mice areintravenously dosed as on day 1. On day 5, mice are weighed, terminatedand blood are collected by heart puncture for Complete Blood Count (CBCanalysis) (plasma-EDTA) and cytokine examination (serum).

CBC and Cytokine Analysis:

Complete blood counts are analyzed from blood draws preceding injections(day−2) and 24 hours after the final injection (day 5). CBC values areassessed for total white blood cell counts and overall red blood cellmorphology. White blood cells are further characterized by total andfractional percentage of neutrophils, lymphocytes, monocytes,eosinophils, & basophils. Red blood cell breakdown included measurementsof hemoglobin (dL), hematocrit (%), mean corpuscular volume (fL), meancorpuscular hemoglobin, mean corpuscular hemoglobin concentration (%),and total platelet count (10³/μL). CBC analysis is performed by AntechDiagnostics (Fishers, Ind.).

Circulating cytokine levels are examined at 4 hours post-injection(day 1) and 24 hours after the final injection (day 5). Serum isisolated, snap frozen and sent to Rules Based Medicine (Austin, Tex.)for multi-analyte profiling. Serum samples are analyzed using theRodentMap panel encompassing 59 unique biomarkers including Apo A-1,CD40, CD40-L, CRP, ET-1, eotaxin, EGF, Factor VII, fibrinogen, FGF-9,FGF-basic, GST-α, GCP-2, GM-CSF, KC/GROα, haptoglobin, IgA, IFNγ, IP-10,IL-1α, IL-1β, IL-10, IL-11, IL-12p70, Il-17A, IL-18, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, LIF, lymphotactin, M-CSF-1, MIP-1α, MIP-1β, MIP-1γ,MIP-2, MIP-3β, MDC, MMP-9, MCP-1, MCP-3, MCP-5, MPO, myoglobin, SAP,SGOT, SCF, RANTES, TPO, tissue factor, TIMP-1, TNF-α, VCAM-1, VEGF-A,and vWF. A change in cytokine levels was counted as a hit if thecytokine increased by at least 2-fold or decreased by at least 50%compared to vehicle controls.

Example 1 Identification of Proteolytic Fragments and Products ofAlternative Splicing from AARSs Using Protein Topography and MigrationAnalysis Platform

To identify AARS fragments from cell lines, conditioned media andtissues, samples are prepared in the following ways:

Mouse Macrophage (RAW 264.7), Cytosol and Conditioned Media:

Cells are treated with serum free DMEM media at a density of 15×10⁶cells/flasks. After 48 hours conditioned media and cell pellets arecollected and processed. 200 μg of protein from secreted and cytosolicproteomic fractions are separated by SDS-PAGE and gel slices areprepared for analysis by mass spectrometry.

Mouse Pancreas Tissue:

The pancreas from three mice are chopped, dounce homogenized, andsonicated in PBS with protease inhibitors. Cytosolic proteome isisolated by centrifugation and 200 μg of protein is separated bySDS-PAGE and gel slices are prepared for analysis by mass spectrometry.

Mouse Liver Tissue:

Three mouse livers are chopped, dounce homogenized, and sonicated in PBSwith protease inhibitors. Cytosolic proteome is isolated bycentrifugation and 200 μg of protein is separated by SDS-PAGE and gelslices are prepared for analysis by mass spectrometry.

In-gel digests are analyzed by LTQ XL ion trap mass spectrometer(ThermoFisher) equipped with ultimate 3000 μLC system (Dionex). Thesamples are first loaded on PepTrap (michrom) for 10 min with 5%Acetonitrile in 0.1% formic acid using Dionex autosampler. Then thesamples are analyzed with a 100 μm (inner diameter) fused silicacapillary column containing 10 cm of C18 resin (michrom). Peptides areeluted from the column into mass spectrometer with a flow rate of0.45p1/min using a linear gradient of 5-33.5% acetonitrile in 0.1%formic acid within 110 min.

LTQ is operated in data-dependent scanning mode such that one full MSscan is followed by seven MS/MS scans of the seven most abundant ions.Dynamic exclusion is enabled with repeat count equals to 1, repeatduration equals to 20 seconds, exclusion list size is 300 and exclusionduration is 60 seconds.

After LC-MS/MS analysis, the raw data is searched withBioWorks3.3.1(SEQUEST) using a concatenated target/decoy variant of themouse IPI database. The SEQUEST data are filtered and sorted withDTASelect. Table 1 shows sequences identified in this way.

Example 2 Identification of Splice Variants Using Deep Sequencing

Splice variants of the aminoacyl tRNA synthetase are identified usinghigh throughput sequencing of cDNA libraries enriched for aminoacyl tRNAsynthetase transcripts. The cDNA templates are prepared from total RNAextracts of tissues such as human adult and fetal brains and enrichedfor aminoacyl tRNA synthetase transcripts by using primer sequencesspecific for all annotated exons of all annotated human aminoacyl tRNAsynthetases and their associated proteins.

Human Total RNAs are obtained from Clontech. For cell line and mousetissue samples, total RNAs are extracted using RNA Extract II Kit (MN).Genomic DNA is digested in the total RNA samples by DNAase I. To obtainmature messenger RNAs (mRNAs), the RNA samples are enriched twice bybinding polyA+ RNA and digestion of RNA without 5′-cap by 5′-phosphatedependent exonuclease. Complementary DNA (cDNA) is synthesized frommature RNAs using primers that anneal to exon sequences of aminoacyltRNA synthetase genes. A transcriptome enriched for aminoacyl tRNAsynthetase genes is amplified by multiplex PCR using the aminoacyl tRNAsynthetase-exon specific cDNA and different combinations of aminoacyltRNA synthetase-exon primers. The double-stranded aminoacyl tRNAsynthetase-enriched transcriptome PCR products are enzymaticallyrepaired at both ends before adding A-overhangs to the 3′ ends of therepaired fragments. Sequencing adaptors and index sequences are thenadded to the aminoacyl tRNA synthetase-enriched transcriptome PCRsproducts to generate cDNA libraries for deep sequencing with Illumina'sMultiplex Sequencing Kit. In brief, the aminoacyl tRNAsynthetase-enriched transcriptome PCR products with 3′-A overhangs areligated to the InPE adaptor oligonucleotides provided in the kits. Indexsequences are added to the PCR products with InPE adaptors. To obtainenough DNA fragments for deep sequencing, the PCR products with indexsequences are further amplified by PCR. Aminoacyl tRNAsynthetase-enriched cDNA libraries with different indexes are pooled andsequenced using an Illumina DNA sequencing machine to get 50 base pairend reads. Sequencing reads are mapped to human or mouse genome foridentification of alternative splicing events. “Splicemap” software(available for public download athttp://www-stat.stanford.edu/˜kinfai/SpliceMap/) is used to identifysplice junctions.

Deep sequencing of these cDNAs are performed to generate about 1 millionsequencing reads of about 50 nucleotides in length. The sequencesspecific for exons of the aminoacyl tRNA synthetases are queried againstannotated exon junctions and new exon junctions are identified asalternative splicing events.

The columns in Table 2 labeled “5′ exon” and “3′exon” indicate, whenpresent, which exons are fused together in the cDNA sequence. Table 2shows sequences that were identified for alternative splice events,transcripts containing such splice events, and the polypeptidesexpressed by those transcripts. Alternative splice variants identifiedby deep sequencing are identified in Table 2 as those ones in whichthere are numbers greater than zero in the columns labeled as“Sequencing reads” in the human adult or fetal brain.

Example 3 Identification of AARS Polypeptides Using Bioinformatics

AARS protein fragments (resectin or appendacrine peptides) areidentified using bioinformatics. Amino acid sequences of the full lengthhuman aminoacyl tRNA synthetase are aligned with the full length aminoacid sequence of its ortholog from the bacterium Escherichia coli usinga program such as FASTA (available at the websitehttp://fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi) or the BLASTPprogram from the NCBI (available at the websitehttp://www.ncbi.nlm.nih.gov/blast/Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthom).Resectin sequences from the human proteins are identified as sequencescovering regions where there are gaps in the bacterial sequence in thealignment, or regions with low homology between the two species. Thepeptide, and corresponding DNA sequences in Table 3 include examplesidentified in this way.

Example 4 Differential Expression of AARS Polypeptides Identified byMass Spectrometry

The PROTOMAP technique is used as described in Example 1 to compare thedifferential expression of tyrosyl tRNA synthetases in differenttissues/cell types (refer to Table 1, for sequences and comparisons):Aminoacyl-tRNA synthetase resectin expression is compared between mouseliver tissue and mouse pancreas tissue. Aminoacyl-tRNA synthetaseresectin expression is compared between cytosol of RAW264.7 andconditioned media from RAW264.7 cells harvested after 48 hours of serumstarvation.

Example 5 Differential Expression of AARS Polypeptides Identified byDeep Sequencing

To test for differential expression of spice events, the deep sequencingis done for cDNAs prepared from different tissues.

Expression of specific alternative splice events for aminoacyl tRNAsynthetases is unexpected and indicates biological importance. Thevariation in relative number of reads seen in the deep sequencing ofdifferent transcriptome samples indicates that alternative splice eventsof aminoacyl tRNA synthetases are differentially regulated and not justartifacts due to sample handling.

Example 6 Antibody Screening

To facilitate the discovery of antibodies displaying preferentialbinding to specific aminoacyl tRNA synthetase fragments (e.g., ≧10-foldhigher affinity when compared to the parental full length enzyme), ahuman antibody phage display library is screened by AbD Serotec (adivision of MORPHOSYS™, Martinsried/Planegg, Germany) using affinityenrichment techniques (panning). Antibodies enriched after multiplerounds of screening with the aminoacyl tRNA synthetase fragments aresubsequently characterized by ELISA for reactivity to the fragments, andto the parental, full length enzyme. Clones demonstrating preferentialbinding (e.g., ≧10-fold higher affinity) to the aminoacyl tRNAsynthetase fragments are further characterized.

If the necessary specificity is not achieved at the end of this process,subtraction strategies, such as pre-adsorption steps with the fulllength enzyme and/or counter-screening, are used to eliminate crossreacting antibodies and drive the selection process towards the uniqueepitope(s) on the aminoacyl tRNA synthetase fragments.

Example 7 Identification of Splice Variants Using Systematic PCR

cDNA templates for PCR reactions are reverse transcribed from total RNAextracts of tissues or cells (e.g., human brain, IMR-32 and HEK293T).PCR reactions are performed using aminoacyl tRNA synthetase specificprimers, pairing a forward primer (FP1) designed to anneal to the 5′untranslated region or exons in the 5′ half of the gene with a reverseprimer (RP1) designed to anneal to exons in the 3′ half of the gene orthe 3′UTR. Amplified DNA products are analyzed by agarose gelelectrophoresis to identify PCR products that are a different size thenthe fragment amplified from the canonical transcripts. These differentPCR products are excised and purified from the gel and ligated into astandard cloning vector for DNA sequence analysis. Alternative splicingvariants are identified as different sequences from the canonicaltranscripts. Splice variants identified by this systematic PCR approachare shown in Table 2.

Example 8 Codon Optimization of Selected AARS Polynucleotides

Representative AARS polypeptides (summarized in Table E2) are selectedfor further biochemical, biophysical and functional characterizationbased on one or more of the following criteria, i) the identification ofAARS polypeptide proteolytic fragments, ii) the identification of AARSpolypeptide splice variants, iii) the identification of AARSpolypeptides by bioinformatic analysis, iv) evidence of differentialexpression of specific AARS polypeptides, v) the domain structure of theAARS protein, vi) the size of the AARS polypeptide, and vii) theminimization of similar duplicative sequences.

TABLE E2 Summary of AARS Polypeptides Selected for Codon Optimizationand Bacterial Expression AARS SEQ. ID Nos. Cloning/ Poly- for EpitopeSEQ. ID. Nos. synthesis peptide Tagged AARS for AARS Poly- Residues ofLocation of method Name polypeptides nucleotides AARS protein epitopetag used TyrRS1^(C1) SEQ. ID. NO. 58 SEQ. ID. NO. 66 390-528 N-terminal2 TyrRS1^(C1) SEQ. ID. NO. 59 SEQ. ID. NO. 66 390-528 C-terminal 2TyrRS1^(C2) SEQ. ID. NO. 60 SEQ. ID. NO. 67 340-528 N-terminal 2TyrRS1^(C2) SEQ. ID. NO. 61 SEQ. ID. NO. 67 340-528 C-terminal 2TyrRS1^(C4) SEQ. ID. NO. 62 SEQ. ID. NO. 68 1-19 + 303-528 N-terminal 2TyrRS1^(C4) SEQ. ID. NO. 63 SEQ. ID. NO. 68 1-19 + 303-528 C-terminal 2TyrRS1^(C5) SEQ. ID. NO. 64 SEQ. ID. NO. 69 1-19 + 171-528 N-terminal 2TyrRS1^(C5) SEQ. ID. NO. 65 SEQ. ID. NO. 69 1-19 + 171-528 C-terminal 2

Polynucleotides encoding the selected AARS polypeptides listed in TableE2, along with the appropriate N or C-terminal epitope tag, aresynthesized and cloned as described in the General Materials and Methodssection using the gene synthesis methodology listed in Table E2.

Example 9 Small Scale Bacterial Expression and Purification

The AARS polypeptides listed in Table E2 are expressed in E. coli. asdescribed in the General Materials and Methods section. The relativeexpression of soluble and inclusion body localized AARS polypeptides issummarized in Table E3 below.

TABLE E3 Summary of AARS Polypeptide Bacterial ExpressionCharacteristics Amount of Protein Recovered Amount of Relative Locationof from Protein Expression AARS Epitope Soluble Recovered from inInclusion Polypeptide Tag Fraction Inclusion Bodies Bodies TyrRS1^(C1)N-terminal + +++ M TyrRS1^(C1) C-terminal + + L TyrRS1^(C2) N-terminal+++++ + L TyrRS1^(C2) C-terminal +++++ + L TyrRS1^(C4) N-terminal ++++ +L TyrRS1^(C4) C-terminal + + L TyrRS1^(C5) N-terminal +++ ++ MTyrRS1^(C5) C-terminal + + L “+” represents 0-1 mg/L AARS polypeptideexpression; “++” represents 1-5 mg/L AARS polypeptide expression; “+++”represents 5-10 mg/L AARS polypeptide expression; “++++” represents10-15 mg/L AARS polypeptide expression; “+++++” represents ≧15 mg/L AARSpolypeptide expression; L: undetectable to low expression levels; M:medium expression levels;

Surprisingly, the protein expression data demonstrates the existence ofat least two protein domains, or alternatively spliced variants thatexhibit high level expression of soluble protein when expressed in E.coli. Specifically the data demonstrates that the AARS polypeptidesTyrRS1^(C2), (amino acids 340-528), and TyrRS1^(C4) (amino acids303-528), define the boundary of a first novel protein domain that ishighly expressed in E. coli; and the alternatively spliced variantTyrRS1^(C5), (amino acids 1-19+171-528), defines the sequence of asecond novel protein domain that is highly expressed in E. coli.

Example 10 Large Scale Production of AARS Polypeptides

Representative AARS polypeptides are prepared in larger amounts toenable further functional and biophysical characterization. The AARSpolypeptides listed in Table E4 are expressed in E. coli in large scaleculture as described in the General Materials and Methods section. Theyields, and specified biophysical characteristics, for each expressedsoluble protein are summarized below in Table E4.

TABLE E4 Summary of representative AARS Polypeptides yield andbiophysical characterization Working stock Stability AARS Location ofYield Purity Endotoxin Molecular concentration [percent AggregationPolypeptide Epitope Tag [mg/L] ⁽¹⁾ [%] [EU/mg] Weight [mg/ml] recovery]⁽²⁾ [AUC] TyrRS1^(C2) C-terminal 12.5 95 0.8 C: 23,597 17.1 98% +++ D:23,468 ⁽⁴⁾ TyrRS1^(C2) N-terminal 11.2 90 2.0 C: 23,597 21.7 75% ++++ D:23,599 TyrRS1^(C4) N-terminal 5.4 85 2.8 C: 29,606 20.7 85% ++++ D:29,609 TyrRS1^(C5) N-terminal 5.0 80 5.2 C: 44,612 19.1 54% +++++ D:44,615, 44,665, 44,696, 89,257 Notes ⁽¹⁾ Yield determined by measuringprotein recovery after last purification step ⁽²⁾ Determined as percentrecovery of non aggregated material after 1 week at 25° C. ⁽⁴⁾ Likely torepresent MW without N-terminal methionine C: Calculated D: Determinedmolecular weight(s) ND: Not Determined “+” represents less than 1% highmolecular weight aggregates “++” represents less than 3% high molecularweight aggregates “+++” represents less than 6% high molecular weightaggregates “++++” represents less than 10% high molecular weightaggregates “+++++” represents more than 10% high molecular weightaggregates

The results from these studies establish that representative AARSproteins from the TyrRS1^(C2), TyrRS1^(C4) and TyrRS1^(C5) families ofAARS proteins, exhibit reasonable initial protein expression yields andsolubility characteristics.

Example 11 Transcriptional Profiling of Representative AARS Polypeptides

To test for the ability of the AARS polypeptides to modulate geneexpression, selected AARS polypeptides were incubated with Mesenchymalstems cells or human skeletal muscle cells for the times and at theconcentrations shown in Table E5.

TABLE E5 Transcriptional profiling of representative AARS Polypeptidesin Mesenchymal Stem Cells (MSC) or Human Skeletal Muscle Cells (HSkMC)Cell type and Exposure Time Test Sample Description Concen- MSC MSCHSkMC HSkMC AARS Location of tration 24 72 24 72 Polypeptides EpitopeTag nM hours hours hours hours TyrRS1^(C1) N-terminal 300 N/A N/A 2 1TyrRS1^(C1) C-terminal 300 0 N/A 0 0 TyrRS1^(C1) N-terminal 100 N/A N/A2 0 TyrRS1^(C1) C-terminal 100 0 N/A 0 0 TyrRS1^(C1) N-terminal 33 N/AN/A 2 0 TyrRS1^(C1) C-terminal 33 0 N/A 0 0 TyrRS1^(C1) N-terminal 11N/A N/A 0 0 TyrRS1^(C1) C-terminal 11 0 N/A 0 0 TyrRS1^(C2) N-terminal300 1 N/A 13 27 TyrRS1^(C2) C-terminal 300 36 N/A 2 5 TyrRS1^(C2)N-terminal 100 1 N/A 1 3 TyrRS1^(C2) C-terminal 100 14 N/A 1 0TyrRS1^(C2) N-terminal 33 3 N/A 1 2 TyrRS1^(C2) C-terminal 33 13 N/A 2 0TyrRS1^(C2) N-terminal 11 1 N/A 2 1 TyrRS1^(C2) C-terminal 11 4 N/A 0 0TyrRS1^(C4) N-terminal 300 19 N/A 1 1 TyrRS1^(C4) C-terminal 300 0 N/A 00 TyrRS1^(C4) N-terminal 100 17 N/A 1 0 TyrRS1^(C4) C-terminal 100 0 N/A0 0 TyrRS1^(C4) N-terminal 33 0 N/A 0 0 TyrRS1^(C4) C-terminal 33 0 N/A0 0 TyrRS1^(C4) N-terminal 11 1 N/A 0 1 TyrRS1^(C4) C-terminal 11 0 N/A0 0 TyrRS1^(C5) N-terminal 300 21 N/A 1 0 TyrRS1^(C5) C-terminal 300 0N/A 0 0 TyrRS1^(C5) N-terminal 100 7 N/A 1 0 TyrRS1^(C5) C-terminal 1000 N/A 0 0 TyrRS1^(C5) N-terminal 33 7 N/A 0 1 TyrRS1^(C5) C-terminal 330 N/A 0 0 TyrRS1^(C5) N-terminal 11 5 N/A 0 0 TyrRS1^(C5) C-terminal 110 N/A 0 0 Controls Average across all AARS 3  5 6 7 polypeptidesscreened Osteogenesis cocktail 17 20 N/A N/A Chondrogenesis cocktail 1719 N/A N/A Adipogenesis cocktail N/A N/A N/A N/A SKMC Pos Ctrl N/A N/A 54 Untreated 0 N/A 3 1

In Table E5, the numbers in each column represent the number of geneswhich were modulated, either positively or negatively by at least 4 foldcompared to the control samples as described in the general methodssection. The data shows that specific forms of the AARS polypeptidestested have the surprising ability to regulate the transcription, andhence potentially modulate the developmental fate or differentiationstatus when added to either Mesenchymal Stem Cells (MSC) and/or HumanSkeletal Muscle Cells (HSkMC). Shaded cells with bolded numbers in thetable represent examples where the AARS polypeptide exhibits asignificant impact on the regulation of gene transcription in the celllines and times indicated in the table.

It is concluded that TyrRS1^(C2), TyrRS1^(C4), and TyrRS1^(C5), appearto be major regulators of Mesenchymal Stem Cell and/or human skeletalmuscle cell gene expression.

Example 12 Functional Profiling of AARS Polypeptides

To test for the ability of the AARS polypeptides to modulate a range ofphenotypic processes, selected AARS polypeptides were incubated with thecell types, and the conditions provided in the general methods section,and Tables E5 and E6.

TABLE E6 Key to Assays and criteria for indicating a hit Proliferationassays Assay Source and cell type Number Human megakaryocytic leukemiacells/Mo7e A1 Human acute promyelocytic leukemia cells/HL60 A2 Humanlymphoblast (cancer cell line)/RPMI8226 A3 Human mesenchymal stemcells/hMSC A4 Human astrocytes A5 Human bone marrow aspirate cells/BoneMarrow Cells A6 Human bone marrow aspirate cells/Bone Marrow Cells (LongTerm A7 Culture) Human Synoviocyte/HFLS-SynRA A8 Human pre-adipocytecells/hPAD A9 Human pulmonary artery smooth muscle cell/hPASMC A10 Humanskeletal muscle cell/hSKMC A11 Data analysis for proliferation assayswas performed by dividing the numerical value in the assay well by theaverage PBS value for the assay plate. AARS polypeptides were consideredto be proliferative if the measured value was greater than 3 SD awayfrom the PBS value in the positive direction. A tRNA synthetase derivedAARS polypeptide was considered to be cytotoxic if greater than 3 SDaway from the PBS value in the negative direction. A cytotoxic compoundwas utilized as a negative control and the average value for this wasalways greater than 3 SD away from PBS average value. Assay AssayDescription Number Cellular differentiation and phenotype assays Humanhepatocyte (HepG2C3a cells) acetylated LDL uptake B1 Data analysis forac-LDL uptake assay was performed by dividing the numerical value in theassay well by the average PBS value for the assay plate. AARSpolypeptides were considered to be a modulator of ac-LDL uptake if themeasured value was greater than 2 SD away from the PBS value in thepositive or negative direction. A visual check to confirm plate readerresults was made using a fluorescent microscope. Human Neutrophil assaysNeutrophil Elastase C1 Neutrophil oxidative burst (agonist) C2Neutrophil oxidative burst (antagonist) C3 Data analysis for neutrophilassays was performed by dividing the numerical value in the assay wellby the average PBS value for the assay plate. AARS polypeptides wereconsidered to be a modulator of neutrophil elastase production oroxidative burst biology if the measured value was greater than 2 SD awayfrom the PBS value in the positive or negative direction. Modulation ofToll-like receptors (TLR) TLR activation in RAW BLUE cells D1 TLRantagonism in RAW BLUE cells D2 Activation of hTLR2 D3 Activation ofhTLR4 D4 Data analysis for TLR modulation assays was performed bydividing the numerical value in the assay well by the average PBS valuefor the assay plate. AARS polypeptides were considered to be a modulatorof TLR specific biology if the measured value was greater than 3 SD awayfrom the PBS value in the positive or negative direction. Positivecontrols, including LPS and detection reagent were always significantlydistinct and >3 SD from PBS average value. Cytokine Release HumanSynoviocyte cytokine production (IL6 release) E1 Human pulmonary arterysmooth muscle cell (hPASMC) cytokine E2 production (IL6 release) Humanskeletal muscle cell (hSKMC) cytokine production (IL6 release) E3 HumanAstrocyte cytokine production (IL6 release) E4 Whole blood IL6 releaseE5 Human pulmonary artery smooth muscle cell (hPASMC) cytokine E6production (IL8release) 72 h Incubation IL8 production Human Synoviocytecytokine production (IL8 release) E7 Human pulmonary artery smoothmuscle cell (hPASMC) cytokine E8 production (IL8release) Human skeletalmuscle cell (hSKMC) cytokine production (IL8 release) E9 Human Astrocytecytokine production (IL8 release) E10 Human hepatocyte (HepG2C3a cells)IL8 release E11 Human acute promyelocytic leukemia cells/HL60 (IL8release) E12 Human lymphoblast (cancer cell line)/RPMI8226 (IL8 Release)E13 TNF alpha production Human Synoviocyte cytokine production (TNFalpha release) E14 Whole blood TNF alpha release E15 IL10 Release Humanacute promyelocytic leukemia cells/HL60 IL10 release E16 Human PrimaryBlood Mononuclear cells (IL10 Release) E17 Data analysis for cytokinerelease assays was performed by dividing the numerical value in theassay well by the average PBS value for the assay plate. AARSpolypeptides were considered to be a modulator of cytokine production orcytokine related biology if the measured value was greater than 2 SDaway from the PBS value in the positive or negative direction. A proteinstandard (specific to each assay kit) was run on every plate to insuregood assay quality. Only assays with protein standard curves that had anR² value of > than 0.9 were chosen for data analysis. Cell Adhesion andChemotaxis Monocyte THP 1/Human umbilical vein endothelial cell (HUVEC)cell F1 adhesion Human hepatocyte (HepG2C3a cells) (ICAM release) F2Human lung microvascular endothelial cell (HLMVEC) cell adhesion F3regulation (ICAM release) Human umbilical vein endothelial cell (HUVEC)cell adhesion regulation F4 (VCAM release) Human mesenchymal stem cell(hMSC) cell adhesion regulation (VCAM F5 release) Human skeletal musclecell (hSKMC) cell adhesion regulation (VCAM F6 release) Human pulmonaryartery smooth muscle cell (hPASMC) cell adhesion F7 regulation (VCAMrelease) Data analysis for cell adhesion regulation assays was performedby dividing the numerical value in the assay well by the average PBSvalue for the assay plate. AARS polypeptides were considered to be amodulator of cell adhesion or a regulator of biology related to celladhesion if a value of greater than 2 SD away from the PBS value in thepositive or negative direction was obtained. In the case of the ELISAassays, a protein standard (specific to each assay kit) was run on everyplate to insure good assay quality. Only assays with protein standardcurves that had an R² value of > than 0.9 were chosen for data analysis.Cellular Differentiation Human pre-adipocyte (hPAD) cell differentiationG1 Human skeletal muscle (hSKMC) cell differentiation G2 Humanmesenchymal stem (hMSC) cell differentiation G3 Human pulmonary arterysmooth muscle cell (hPASMC) differentiation G4 Data analysis forcellular differentiation assays was performed by dividing the numericalvalue in the assay well by the average PBS value for the assay plate.Differentiation assays were scored based upon fluorescent intensity ofparticular antibodies as described in the methods section. AARSpolypeptides were considered to be a modulator of cellulardifferentiation if an intensity value for a specific marker ofdifferentiation was greater than 2 SD away from the PBS value in thepositive or negative direction in a given treated well. For the hSKMCanalysis, digital photos were taken of all wells and photos were scoredin a blinded fashion by three people using a 4 point scoring systemwhere a score of “4” indicated intense skeletal muscle actin stainingand obvious myotube formation and a score of “1” indicated a lack of anydifferentiation or a suppression of differentiation. The average valuefrom visual scoring was used and only wells with an average value of >3were considered hits. Differentiation control treated wells in thisassay typically scored >2, while PBS treated wells scored <2. CellBinding PBMC H1 Primary T cell H2 Primary B cell H3 Primary Monocyte H4HepG2 H5 3T3L1 H6 C2C12 H7 THP1 H8 Jurkat H9 Raji H10 AARS polypeptideswere considered to be binding to a particular cell type if the mean cellbound fluorescence intensity was greater than 2 SD away from the reagentcontrol values for that cell type.

TABLE E7 Results of Functional Profiling studies of AARS PolypeptidesAARS Location of Concentration Polypeptides Epitope Tag [nM] Assay HitsTyrRS1^(C1) N-terminal 300 B1 (Ac-LDL Uptake) E7 (Cytokine Release) F2,F4 (Cell Adhesion and Chemotaxis) TyrRS1^(C2) N-terminal 300 B1 (Ac-LDLUptake) A7 (Proliferation) E1, E4, E7 (Cytokine Release) F2, F6 (CellAdhesion and Chemotaxis) TyrRS1^(C2) C-terminal 300 A6 (THP1 possiblesuppression of growth), A7 (Proliferation) B1 (Ac-LDL Uptake) D1(Toll-like Receptor Biology) E3, E8, E7(Cytokine Release) F4 (CellAdhesion and Chemotaxis) TyrRS1^(C4) N-terminal 300 A7 (Proliferation)B1 (Ac-LDL Uptake) E1, E4 (Cytokine Release) F2, F4, F6 (Cell Adhesionand Chemotaxis) TyrRS1^(C5) N-terminal 300 B1 (Ac-LDL Uptake), D1(Toll-like Receptor Biology) E6, E7 (Cytokine Release) F1 (Cell Adhesionand Chemotaxis) TyrRS1^(C5) N-terminal 300 B1 (Ac-LDL Uptake), E3(Cytokine Release) F2 (Cell Adhesion and Chemotaxis)

It is concluded that TyrRS1^(C1), TyrRS1^(C2), TyrRS1^(C4) andTyrRS1^(C5) appear to be major regulators of proliferation,differentiation, cytokine release, neutrophil activation, cell adhesionand chemotaxis. Of note is that in many cases, the N and C-terminalfusion proteins have differential patterns of activity in both thetranscriptional profiling experiments, as well as in the phenotypicscreening experiments. This data is consistent with the hypothesis thatfor these AARS polypeptides, the novel biological activity is suppressedwhen the AARS polypeptide is part of the intact tRNA synthetase, ortranslationally fused at either terminus to another protein, but thatthis biological activity is revealed when the AARS polypeptides has afree amino or carboxy terminus.

When viewed in the context of the transcriptional profiling studies, thephenotypic screening data demonstrates that the AARS polypeptidesTyrRS1^(C1), (amino acids 390-528), TyrRS1^(C2) (amino acids 340-528),TyrRS1^(C4) (amino acids 303-528, define the boundary of a first novelprotein domain that is highly active in a broad array of phenotypicscreening assays.

Accordingly it is concluded that AARS polypeptides comprising aminoacids 303-528 of tyrosyl tRNA synthetase define the approximateboundaries (i.e. within about +/−5 amino acids) of a first, highlyactive AARS polypeptide domain, that is i) highly functionally active,ii) can be readily made and produced in E. coli, and iii) exhibitsfavorable protein stability and aggregation characteristics. It will beappreciated by those of skill in the art that any AARS polypeptidescomprising as few as the last 390 amino acids of the tyrosyl tRNAsynthetase, to as large as polypeptides comprising the last 303 aminoacids of tyrosyl tRNA synthetase represent functional equivalents of thespecific AARS polypeptides described.

The phenotypic screening data also demonstrates that the alternativelyspliced AARS polypeptide TyrRS1^(C5), (amino acids 1-19+171-528) definethe boundaries of a second novel protein domain that is highly active ina broad array of phenotypic screening assays. Accordingly it isconcluded that AARS polypeptides comprising amino acids 1-19+171-528 oftyrosyl tRNA synthetase define the approximate boundaries (i.e. withinabout +/−5 amino acids) of a second novel, highly active AARSpolypeptide domain, that is i) highly functionally active, ii) can bereadily made and produced in E. coli, and iii) exhibits favorableprotein stability and aggregation characteristics.

1-132. (canceled)
 133. A therapeutic composition, comprising apharmaceutically-acceptable carrier and an antibody or antigen-bindingfragment thereof that exhibits binding specificity for an isolatedaminoacyl-tRNA synthetase (AARS) polypeptide selected from Table(s) 1-3,or Table(s) 4-6, and Table(s) 7-9, wherein the composition has a purityof at least about 90% on a protein basis and less than about 10 EUendotoxin/mg protein.
 134. The therapeutic composition of claim 133,wherein the AARS polypeptide is selected from Table E7.
 135. Thetherapeutic composition of claim 133, wherein the antibody or antigenbinding fragment thereof is a monoclonal antibody.
 136. The therapeuticcomposition of claim 133, wherein the antibody is a humanized antibody.137. The therapeutic composition of claim 133, wherein the antibody orantigen-binding fragment thereof is an Fv fragment or a single chain Fv(sFv) polypeptide.
 138. The therapeutic composition of claim 133,wherein the composition is a sterile, freeze-dried powder.
 139. Thetherapeutic composition of claim 133, wherein the composition is asterile injectable solution.
 140. The therapeutic composition of claim133, wherein the composition is buffered and comprises an isotonicagent.
 141. The therapeutic composition of claim 133, wherein thecomposition is suitable for intravenous, intramuscular, subcutaneous, orintraperitoneal administration.
 142. The therapeutic composition ofclaim 133, wherein the composition comprises a surfactant.
 143. Thetherapeutic composition of claim 133, wherein the composition has apurity of at least about 95% on a protein basis.
 144. A method ofmodulating a cellular activity of a cell, or protein, comprisingcontacting the cell or protein with a therapeutic composition of claim133.
 145. The method of claim 144, wherein the cellular activity isselected from cell proliferation, cell differentiation, cell signaling,gene transcription, angiogenesis, cell binding, metabolism, cytokineproduction or activity, cytokine receptor activity, and inflammation.146. The method of claim 144, wherein the cell type is selected from thegroup consisting of pre-adipocytes, bone marrow, neutrophils, bloodcells, hepatocytes, astrocytes, mesenchymal stem cells, and skeletalmuscle cells.
 147. The method of claim 144, wherein the cell is in asubject.
 148. The method of claim 147, comprising treating the subject,wherein the subject has a condition associated with a neoplasticdisease, an immune system disease or condition, an inflammatorydisorder, an infectious disease, a metabolic disease,neuronal/neurological disease, a muscular/cardiovascular disease, adisease associated with aberrant hematopoiesis, a disease associatedwith aberrant angiogenesis, or a disease associated with aberrant cellsurvival.
 149. A method of reducing or ameliorating a symptom of adisease mediated by a mutation, inappropriate subcellular association,over-expression, or subcellular distribution of tyrosyl-tRNA synthetase(TyrRS), comprising administering to a subject a therapeutic compositionof claim 133.